article_id
stringlengths 6
9
| article_text
stringlengths 122
2.02M
| document_type
stringclasses 4
values | domain
stringclasses 3
values | language
stringclasses 27
values | language_score
float64 0
1
|
|---|---|---|---|---|---|
PMC11695210 | Section title: INTRODUCTION Educational score: 3.929046630859375 Domain: biomedical Document type: Study Language: en Anthracyclines are often used in the treatment of breast cancer. 1 Patients with breast cancer receiving anthracyclines are at high risk of cancer therapy–related cardiac dysfunction (CTRCD), which reduces adherence to anti‐cancer treatments and may ultimately decrease the overall survival rates. 2 Therefore, risk stratification of CTRCD before anthracycline chemotherapy is important for breast cancer patients. Section title: INTRODUCTION Educational score: 4.034027576446533 Domain: biomedical Document type: Review Language: en Baseline risk stratification of CTRCD is a challenging task in breast cancer patients with anthracycline administration. 3 These clinically known cardiovascular disease (CVD) risk factors (e.g., age, body mass index, diabetes, hypertension, dyslipidemia, and smoking) failed to accurately stratify CTRCD risk owing to ignoring the differences in cancer treatment modalities and cardiac structural and functional subtypes. 4 , 5 According to the 2022 European Society of Cardiology guidelines on cardio‐oncology, the Heart Failure Association‐International Cardio‐Oncology Society (HFA‐ICOS) score including previous CVDs, cardiac biomarkers, demographic and cardiovascular risk factors, previous cardiotoxic cancer treatment, and lifestyle risk factors is recommended to baseline risk stratification of CTRCD in breast patients receiving anthracyclines. 6 However, this recommendation is derived from level of evidence B or C. 7 Therefore, validation of the current HFA‐ICOS score and new parameters that can predict risk of CTRCD are priorities in breast cancer patients receiving anthracyclines. Section title: INTRODUCTION Educational score: 4.066875457763672 Domain: biomedical Document type: Study Language: en Cardiac magnetic resonance (CMR) cine imaging, which is a basic part of the CMR examination, has been established as a noninvasive and noncontrast modality for assessing the structure and function of the heart. 8 Left ventricular ejection fraction (LVEF) and global longitudinal strain (GLS) derived from CMR cine images are considered as risk predictors of CTRCD in breast cancer patients receiving anthracyclines. 9 However, the accuracy of LVEF and GLS at baseline for risk stratifying CTRCD remains controversial, because LVEF often identifies only irreversible CTRCD, and GLS is highly dependent on the load and chamber size of left ventricle. 10 , 11 Section title: INTRODUCTION Educational score: 4.047896385192871 Domain: biomedical Document type: Study Language: en The left ventricular trabecular fractal dimension (LVTFD) derived from CMR cine sequence reflects myocardial trabecular complexity and has been identified as a new and important parameter for the determinant of cardiac performance and has a causal relationship with the risk of CVD. 12 Recent studies have reported that LVTFD is a new biomarker of cardiac involvement in Fabry disease 13 and risk stratification of adverse cardiovascular events in hypertrophic cardiomyopathy. 14 However, it is unclear whether LVTFD can be used as a new parameter to risk stratification of CTRCD and whether it improves the performance of HFA‐ICOS score for risk stratification of CTRCD in breast cancer patients receiving anthracyclines. Section title: INTRODUCTION Educational score: 4.0739288330078125 Domain: biomedical Document type: Study Language: en Therefore, this study aimed to (i) validate the current HFA‐ICOS score for baseline risk stratification of CTRCD and (ii) derive and validate an LVTFD‐based score for stratifying CTRCD risk in comparison to HFA‐ICOS score in breast cancer patients receiving anthracyclines. Section title: Study population characteristics Educational score: 4.108782768249512 Domain: biomedical Document type: Study Language: en The flowchart of this study is shown in Figure 1 . Fourteen participants were excluded because of anti‐tumor treatment before CMR ( n = 4), contraindication to magnetic resonance imaging ( n = 5), and serious artifacts of CMR ( n = 5). Finally, 370 participants were enrolled and they were divided into the derivation cohort ( n = 222) and the validation cohort ( n = 148). CTRCD occurred in 73 (73/370, 19.8%) participants (44 [44/222, 20.0%] in derivation cohort and 29 [29/148, 19.6%] in validation cohort). The median CTRCD‐free survival time was 18.0 [interquartile range (IQR), 12–24] months. Section title: Study population characteristics Educational score: 2.4412128925323486 Domain: biomedical Document type: Study Language: en The demographic and clinical characteristics and CMR data are shown in Table 1 . There were no differences in baseline demographic and clinical characteristics except for hypertension and previous CVD between those with and without CTRCD in both derivation and validation cohorts. Section title: Study population characteristics Educational score: 4.142714977264404 Domain: biomedical Document type: Study Language: en There were excellent intra‐ and interobserver reproducibility in LVEF, left ventricular end‐diastolic volume (LVEDV), end‐systolic volume (LVESV), and myocardial mass (LVMASS), GLS, global circumferential strain (GCS), global radial strain (GRS), global fractal dimension (FD), maximal basal FD, mean basal FD, maximal apical FD, and mean apical FD, with the interclass correlation coefficient (ICC) value ranging from 0.846 to 0.957. Detailed ICC values for all variables are presented in Table S1 . Pearson's correlation analysis showed that all left ventricular FDs were not associated with ventricular function and mass parameters, as shown in Tables S2 and S3 . Section title: Study population characteristics Educational score: 4.055055618286133 Domain: biomedical Document type: Study Language: en In both derivation and validation cohort, the global FD, maximal and mean basal FD, and maximal and mean apical FD were higher in participants with CTRCD than those in participants without CTRCD (all p < 0.05). There were no differences in GLS, GCS, and GRS between those with and without CTRCD in both derivation and validation cohorts (all p > 0.05). Section title: Derivation of LVTFD‐based score Educational score: 4.074910640716553 Domain: biomedical Document type: Study Language: en As shown in Figure 2 , the age, hypertension, previous CVD, and maximal apical FD (adjusted hazard ratios: 2.246, 1.589, 2.372, and 2.630, respectively) were selected as components for the LVTFD‐based score. We considered a woman aged <65 years without hypertension or previous CVD and with maximal apical FD <1.272 as the reference, she would have a LVTFD‐based score of 0. Individual risk factors contributed 1 or 2 points to the LVTFD‐based score. Individuals with hypertension were assigned 1 point and individuals aged ≥65 years or those with previous CVD or maximal apical FD ≥ 1.272 were assigned 2 points. The subjects with LVTFD‐based scores of 0–1, 2–3, and ≥ 4 points were divided into low‐, moderate‐, and high‐risk groups, respectively. Section title: Comparison of HFA‐ICOS score and LVTFD‐based score performance for the baseline risk stratification of CTRCD Educational score: 4.080664157867432 Domain: biomedical Document type: Study Language: en As shown in Table 2 , there were significant differences in HFA‐ICOS score and LVTFD‐based score between the participants with and without CTRCD in both derivation and validation cohorts (all p < 0.001). In derivation cohort, 84.3%, 11.8%, and 3.9% of participants without CTRCD were classified as low‐, moderate‐, and high‐risk and 50.0%, 31.8%, and 18.2% of participants with CTRCD were classified as low‐, moderate‐, and high‐risk according to the HFA‐ICOS score. In derivation cohort, 59.6%, 36.5%, and 3.9% of participants without CTRCD were classified as low‐, moderate‐, and high‐risk, and 27.3%, 50.0%, and 22.7% of participants with CTRCD were classified as low‐, moderate‐, and high‐risk according to the LVTFD‐based score. Similar results were found in validation cohort. Section title: Comparison of HFA‐ICOS score and LVTFD‐based score performance for the baseline risk stratification of CTRCD Educational score: 4.079678535461426 Domain: biomedical Document type: Study Language: en As shown in Table 3 , the C‐indices of LVTFD‐based score were higher than those of HFA‐ICOS score for the baseline risk stratification of CTRCD (0.834 vs. 0.642, p < 0.001 and 0.834 vs. 0.633, p < 0.001, respectively, in the derivation and validation cohorts).The C‐indices of LVTFD‐based score were higher than those of the combination of age, hypertension, and previous CVD (0.834 vs. 0.671, p < 0.001 and 0.834 vs. 0.674, p < 0.001, respectively, in both derivation and validation cohorts). Section title: Comparison of HFA‐ICOS score and LVTFD‐based score performance for the baseline risk stratification of CTRCD Educational score: 4.073846340179443 Domain: biomedical Document type: Study Language: en Kaplan–Meier survival curves stratified by HFA‐ICOS score for the baseline risk stratification of CTRCD are shown in Figure 3 . The CTRCD‐free survival of high‐risk subjects and moderate‐risk subjects was higher than low‐risk subjects (all p < 0.001), respectively. However, the CTRCD‐free survival showed no difference between high‐ and moderate‐risk subjects in both the derivation and validation cohorts ( p = 0.481 and p = 0.606, respectively). Section title: Comparison of HFA‐ICOS score and LVTFD‐based score performance for the baseline risk stratification of CTRCD Educational score: 4.109498023986816 Domain: biomedical Document type: Study Language: en Kaplan–Meier survival curves analysis stratified by LVTFD‐based score for baseline risk stratification of CTRCD are shown in Figure 3 . The CTRCD‐free survival of high‐risk subjects and moderate‐risk subjects was higher than that of low‐risk subjects, respectively, in both derivation ( p < 0.001, p = 0.005) and validation cohorts ( p < 0.001, p = 0.016). In addition, the CTRCD‐free survival of high‐risk subjects was also significantly higher than that of moderate‐risk subjects in both derivation ( p < 0.001) and validation cohorts ( p = 0.015). Section title: Comparison of HFA‐ICOS score and LVTFD‐based score performance for the baseline risk stratification of CTRCD Educational score: 2.507458448410034 Domain: biomedical Document type: Study Language: en The CTRCD rate at 1 year, 2 years, and the end of follow‐up of different risk subgroups are listed in Table 4 . Section title: DISCUSSION Educational score: 4.036808013916016 Domain: biomedical Document type: Study Language: en In this study, we found that the performance of HFA‐ICOS score for baseline risk stratification of CTRCD was unsatisfactory. Our new LVTFD‐based score integrating clinical risk factors and CMR‐derived LVTFD significantly improved the performance of the baseline risk stratification for CTRCD. Section title: DISCUSSION Educational score: 4.101161956787109 Domain: biomedical Document type: Study Language: en Baseline risk stratification of CTRCD is important in patients with breast cancer receiving anthracycline, because it enables the oncologist to consider cardiotoxicity risk and make personalizing cancer treatment and cardioprotective strategies. 15 The HFA‐ICOS score is considered to determine baseline risk of CTRCD in cancer patients receiving anthracyclines. 16 Our study verified the performance of HFA‐ICOS score for baseline risk stratification of CTRCD, which yielded unsatisfactory C‐indices (0.642 and 0.633, respectively, in derivation and validation cohorts). This result is in line with recent studies validating the HFA‐ICOS score for stratifying CTRCD risk in HER2+ breast cancer [area under the curve (AUC): 0.58 and 0.643]. 17 , 18 The possible explanation is that the HFA‐ICOS score is based on level of evidence B or C and only depends on clinical cardiovascular risk factors. 7 This indicates the need to develop new biomarkers for the baseline risk stratification of CTRCD in breast cancer patients receiving anthracyclines. Section title: DISCUSSION Educational score: 4.120151519775391 Domain: biomedical Document type: Study Language: en The FD of ventricular trabeculae calculated based on CMR cine images is an important biomarker of cardiac function. 19 , 20 Previous studies have shown that the maximal apical FD of ventricular trabeculae is a predictor of cardiac adverse events in patients with pulmonary hypertension and hypertrophic cardiomyopathy. 14 , 21 Interestingly, we found that the maximal apical FD of left ventricular trabeculae was an ideal marker for baseline risk stratification of CTRCD in breast cancer patients receiving anthracyclines. The potential explanation may be that trabecular complexity represented by the FD is determined by cardiac genes and is an important biomarker of individual variation in cardiac efficiency. 12 The maximal apical FD of left ventricular trabeculae can be calculated on the images of basic cine sequence in CMR. 22 With short scanning time and no need for contrast agent, 23 maximal apical FD of left ventricular trabeculae may be recommended for the risk stratification of CTRCD in breast cancer patients receiving anthracyclines. It is conducive to the implementation of the latest cardiac protective treatment. 24 Section title: DISCUSSION Educational score: 4.169048309326172 Domain: biomedical Document type: Study Language: en Our LVTFD‐based score classified 50.0% and 22.7% of participants with CTRCD as moderate‐ and high‐risk, whereas HFA‐ICOS score classified 31.8% and 18.2% of them as moderate‐ and high‐risk in the derivation cohort, and the results were similar in the validation cohort. This indicated that more CTRCD participants can be identified with medium‐high risk using LVTFD‐based score compared with medium‐high risk using HFA‐ICOS score. Possible explanations are as follows: First, the occurrence of CTRCD was attributable not only to clinical risk factors but also to the cardiac phenotype, which determines the heart's ability to tolerate adverse stimuli such as anthracyclines. 25 LVTFD‐based score is calculated based on clinical risk factors including age, hypertension, previous CVD and CMR‐derived cardiac phenotype biomarker, and maximal apical FD of left ventricular trabecular, which is associated with risk of CVD. 12 , 20 , 26 The HFA‐ICOS score is calculated only based on cardiovascular risk factors. Second, both the presence of CTRCD events and their timing were considered in the development of our new scoring system. This LVTFD‐based score classified 36.5% and 35.3% of participants without CTRCD as moderate‐risk, whereas HFA‐ICOS score classified 11.8% and 10.9% of them as moderate‐risk in derivation and validation cohorts, respectively. This indicated that participants with CTRCD classified as moderate‐risk by LVTFD‐based score maybe should undergo more frequent cardiotoxicity monitoring. Section title: DISCUSSION Educational score: 3.9552793502807617 Domain: biomedical Document type: Study Language: en Previous studies showed that the incidence of CTRCD in breast cancer patients who received anthracyclines was 3%–20%. 27 , 28 , 29 In our study, the incidence of CTRCD was 19.7%. There are several possible explanations. First, most patients come from remote rural areas with very low incomes. The anthracyclines received by most participants are doxorubicin, which is cheap and at high risk of cardiotoxicity. Second, the enrolled people are only from one region of a single country, and people from different countries and regions have different abilities to withstand adverse cardiac stimuli like anthracycline chemotherapy. Section title: DISCUSSION Educational score: 3.8837130069732666 Domain: biomedical Document type: Study Language: en There were some limitations in this study. First, the enrolled population in our study is potentially skewed because all participants underwent CMR. Second, the LVTFD‐based score was derived from a single‐center study with a modest sample size. All participants were Asian with very low rate of baseline cardiovascular risk factors, differing considerably from western populations. Additional validations from multiple, international centers, and large samples are needed. Third, the median follow‐up is short in our study (18 months); long‐term follow‐up studies are needed to validate our scoring system. Section title: DISCUSSION Educational score: 4.0177106857299805 Domain: biomedical Document type: Study Language: en In conclusion, LVTFD‐based score is an alternative metric for baseline risk stratification of CTRCD in breast cancer patients receiving anthracyclines. The LVTFD‐based score may be helpful for accurate baseline risk stratification of cardiotoxicity and for individualizing treatment strategies in breast cancer patients receiving anthracyclines. Section title: Study population Educational score: 2.041964530944824 Domain: biomedical Document type: Study Language: en This prospective and single‐center study received approval from the local ethics committee and was conducted following the Declaration of Helsinki. All participants gave written informed consent. Section title: Study population Educational score: 4.024191856384277 Domain: biomedical Document type: Study Language: en Between January 2020 and March 2022, consecutive participants with breast cancer scheduled to receive anthracyclines and underwent CMR cine imaging were enrolled in this prospective study. The inclusion criteria were as follows: (i) pathologically confirmed female breast cancer; (ii) no history of anti‐tumor treatment before CMR examination. The exclusion criteria were as follows: (i) contraindication to CMR and (ii) serious artifacts of CMR. Section title: Study population Educational score: 2.221867084503174 Domain: biomedical Document type: Study Language: en All enrolled participants were randomly divided into the derivation or the validation cohort in a 3:2 ratio. Section title: CMR protocol Educational score: 4.054128170013428 Domain: biomedical Document type: Study Language: en CMR was performed on a 3.0‐T magnet (Ingenia, Philips Healthcare) with a 32‐channel coil array agent. Compressed sensing cine sequences in short‐axis, two‐chamber, three‐chamber, and four‐chamber views were performed with electrocardiogram‐gating and breath‐holding (8‐mm thick with a 10% gap). The detailed parameters of CMR imaging are summarized as follows: readout sequence; balanced steady‐state free precession; slice thickness/gap: 8 mm; repetition time: 3 ms; echo time: 1.48 ms; SENSE factor: 2; phase partial Fourier: off; average: 1; bandwidth: 1645 Hz; flip angle: 45°; field of view: 270 × 270 mm; Voxel size: 1.8 × 1.8 mm; calculated phases: 30. Section title: CMR cine data analysis Educational score: 3.5318663120269775 Domain: biomedical Document type: Study Language: en CMR cine data were analyzed by two radiologists with 5 and 10 years of cardiac imaging experience blinded to the clinical and grouping information on cvi42 software version 5.16 (Circle Cardiovascular Imaging Inc.). Section title: CMR cine data analysis Educational score: 4.002744197845459 Domain: biomedical Document type: Study Language: en In the tissue tracking module, epicardial and endocardial contours were automatically generated and manually corrected if necessary. LVEF, LVEDV, LVESV, LVMASS, GLS, GCS, and GRS were measured. The GLS and GCS values are expressed as absolute numbers. Section title: CMR cine data analysis Educational score: 4.112687587738037 Domain: biomedical Document type: Study Language: en FD analysis was performed by the two radiologists mentioned above using the cvi42 software, prototype5.3.8. Endocardial contour was automatically traced at the end‐diastolic phase of every short‐axis slice and manually adjusted if necessary. Then, the FD values of each slice were automatically calculated. The left ventricular stack was split into apical and basal halves ; the maximum and mean apical and basal FD and global FD were reported as a previous study . 14 The global FD was defined as the mean value of FDs in all left ventricular slices. The mean apical or basal FD was defined as the mean value of all slices of the apex or base of the left ventricle. The maximum apical or basal FD was defined as the maximum value of all slices of the apex or of base the left ventricle. Section title: CTRCD definition and monitoring Educational score: 4.145627021789551 Domain: biomedical Document type: Study Language: en CTRCD was defined as ≥10% reduction in echocardiography measured LVEF to <53% according to the American Society of Echocardiography/European Association of Cardiovascular Imaging. 30 For monitoring CTRCD, the cardiac troponin, natriuretic peptides, and echocardiography were performed every two cycles during anthracycline chemotherapy, every 3 months within 1 year after therapy completion, every 6 months with 1–2 years after therapy completion, and annually more than 2 years after therapy completion. All participants were followed for at least 1 year, the endpoint of follow‐up was April 2023. CTRCD‐free survival time was recorded from the start of anthracyclines to the occurrence of CTRCD or the endpoint of follow‐up. Section title: Derivation of LVTFD‐based score Educational score: 4.079538345336914 Domain: biomedical Document type: Study Language: en Clinical and CMR variables with p < 0.2 or clinically known CVD risk factors (e.g., age, body mass index, diabetes, hypertension, dyslipidemia, and smoking) regardless of p value in univariate analysis were entered into a multivariate Cox proportional hazards model analysis. Variables with p values < 0.10 in the multivariate analysis were then used to construct a risk score. 31 The point of each covariate is equal to its coefficient divided by the coefficient with the smallest absolute value in the model and rounded to an integer. The LVTFD‐based score for a given individual was the total sum of points. The optimal cut‐off values for FD and the LVTFD‐based score for baseline risk stratification of CTRCD were calculated using X‐tile software (version 3.6.1; Yale University). Then, the participants were divided into low‐, moderate‐, and high‐risk subgroups based on the cut‐off value. Section title: HFA‐ICOS score calculation Educational score: 3.641122579574585 Domain: biomedical Document type: Study Language: en Moderate 1 and 2, high‐risk, and very high‐risk factors were defined according to HA‐ICOS score proforma. HFA‐ICOS score was divided into four risk subgroups as follows: low risk, no risk factors, or one Moderate 1 risk factor; moderate risk, moderate risk factors with a total of 2–4 points (Moderate 1 = 1 point; Moderate 2 = 2 points); high‐risk, moderate‐risk factors with a total of ≥5 points or any high‐risk factor and very high‐risk factor. 16 Section title: Statistical analysis Educational score: 4.095485687255859 Domain: biomedical Document type: Study Language: en The ICC was calculated for evaluating the reproducibility of continuous variables. The normality of data was tested with Shapiro–Wilk test. Mean and SD were used to present continuous variables with normal distribution. Median and IQR were used to present continuous variables with non‐normal distribution. Frequencies and proportions were used to present categorical variables. The two‐sample Student's t ‐test or Mann–Whitney U test was used to compare continuous variables, and the chi‐square or Fisher exact test was used to compare categorical variables between participants with and without CTRCD. Univariable and multivariable Cox proportional hazard model was used to access the associated variables with CTRCD. The proportional hazards assumption test hold water. The performance of risk CTRCD was evaluated by Harrell's C‐statistic (C‐index) and was compared using Delong's test. CTRCD‐free survival in three different risk subgroups was compared using Kaplan–Meier curves and log‐rank test. Pairwise comparisons were performed using the Bonferroni correction, with a two‐tailed p value of <0.05/3 indicating a statistical difference. For the remaining statistics, two‐tailed p value of <0.05 indicated a statistical difference. Statistical analyses were performed in R (version 3.6.2; The R Foundation). Section title: AUTHOR CONTRIBUTIONS Educational score: 0.9880476593971252 Domain: other Document type: Other Language: en Conceptualization : Hesong Shen, Jiuquan Zhang, and Qian Xu. Methodology : Chunrong Tu and Yangling Peng. Investigation : Zhiming Miao. Statistical analyses : Yuhang Xie and Rui Yang. Writing—original draft : Hesong Shen. Writing—review and editing : Jiuquan Zhang, Chunrong Tu, and Qian Xu. Funding acquisition : Jiuquan Zhang, Hesong Shen, and Chunrong Tu. Hesong Shen and Jiuquan Zhang had unrestricted access to all data. All authors agreed to submit the manuscript, read, and approve the final draft and take full responsibility of its content. Jiuquan Zhang had final responsibility for the decision to submit for publication. Section title: ETHICS STATEMENT Educational score: 1.0863443613052368 Domain: biomedical Document type: Other Language: en This clinical study was approved by the ethics committee of Chongqing University Cancer Hospital . Section title: CONFLICT OF INTEREST STATEMENT Educational score: 0.8662789463996887 Domain: other Document type: Other Language: en The authors declare no conflicts of interest. | Study | biomedical | en | 0.999995 |
PMC11695213 | Section title: Introduction Educational score: 4.035462856292725 Domain: biomedical Document type: Review Language: en Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a primary liver cancer (PLC) with heterogeneous phenotypes that share common characteristics of both hepatocytic and cholangiocytic differentiation ( 1 ). cHCC-CCA is rare, with reported incidences ranging from 0.4% to 14.2% of PLCs. The World Health Organization (WHO) estimates a similar incidence at 2%–5% of PLCs ( 2 – 4 ). The cHCC-CCA was initially described by Allen and Lisa in 1949, nevertheless, the demographic and clinical features of these tumors remain ambiguous. Section title: Introduction Educational score: 4.152604103088379 Domain: biomedical Document type: Study Language: en Microvascular invasion (MVI) serves as an indicator of tumor invasiveness and is an adverse prognostic factor associated with early disease recurrence and lower survival rates ( 5 – 7 ). MVI is characterized by the infiltration of tumor cells into small blood vessels surrounding the tumor, such as the portal vein and hepatic vein systems, indicating a more aggressive biological behavior. Some scholars posit that MVI is the first step in the development of intrahepatic or systemic metastasis in liver cancer ( 8 , 9 ). Consequently, preoperative prediction of MVI would facilitate treatment planning and enhance prognosis. Hence, some researchers suggest that patients with cHCC-CCA who are predicted to have MVI should undergo anatomical liver resection, expanding the scope of lesion removal to reduce early recurrence rates ( 10 – 12 ). Therefore, early and accurate assessment of MVI has significant implications for treatment decisions and prognosis prediction in cHCC-CCA patients. Unfortunately, the confirmation of MVI mostly depends on histopathological examination of surgical specimens. Currently, there are limited reports on the predictive role of preoperative contrast-enhanced ultrasound (CEUS) in detecting MVI in cHCC-CCA patients ( 13 ). However, CEUS can reflect the blood perfusion of tumor tissue in real time, which has important clinical value in the diagnosis of focal liver lesions. In addition, due to the relatively small sample size, the imaging features of cHCC-CCA on CEUS and its relationship with histopathological features are not well summarized. Hence, the objective of this study is to analyze the prediction of MVI in cHCC-CCA using preoperative CEUS and clinicopathological features. Section title: Patients Educational score: 4.1106767654418945 Domain: biomedical Document type: Study Language: en This retrospective study included 57 cases of cHCC-CCA patients who received medical treatment at Guizhou Provincial People’s Hospital between November 2017 and May 2023, and were confirmed by pathology. The implementation of this study has been approved by the Ethics Committee of our hospital, and informed consent of the subjects has been waived . The inclusion criteria were as follows (1): cHCC-CCA confirmed by surgery and pathology based on the 2019 WHO classification ( 14 ); (2) undergoing CEUS examination within 2 weeks prior to surgery; (3) complete preoperative clinical data available; (4) presence of MVI information in the postoperative histopathological results. The exclusion criteria were: (1) previous anti-cancer treatments such as local therapy or systemic chemotherapy; (2) presence of concurrent malignancies in other sites; (3) lack of preoperative radiological and clinical data. Figure 1 illustrates the patient recruitment process. Section title: CEUS techniques Educational score: 4.055147171020508 Domain: biomedical Document type: Study Language: en The Aixplorer Sxc6-1 ultrasound system (USA, SuperSonic Imagine) and the Mylab-90 color Doppler ultrasound diagnostic device (Esaote C1-8) with a probe frequency of 1-5 MHz were used. The ultrasound contrast agent SonoVue (Bracco, Italy) was utilized. Before use, it was diluted with 5 ml of 0.9% sodium chloride solution, vigorously shaken, and then injected via the superficial vein of the elbow using a bolus injection method with a volume of 1.5-2.2 ml (determined based on the patient’s weight), followed by a flush with 5 ml of 0.9% sodium chloride solution. The procedure adhered to the guidelines of the Chinese 2017 Ultrasound Contrast Imaging Manual ( 15 ). The liver was scanned in the conventional 2D mode to record the number, location, size, borders, internal echoes, and color Doppler blood flow signals of the tumors. Prior to contrast imaging, communication with the patient was conducted to select the optimal position for visualizing the tumor lesions. The contrast agent was injected in the imaging mode, and the timer was started simultaneously. The lesions were continuously monitored for a duration of 5 minutes. If multiple lesions were present, the one with the largest diameter was considered the primary observation target. The contrast images were analyzed and diagnosed by two or more physicians with the rank of associate chief physician or higher. Section title: US and CEUS image analysis Educational score: 4.182634353637695 Domain: biomedical Document type: Study Language: en Two professional hepatologists with at least 5 years of experience in liver CEUS analysis retrospectively reviewed ultrasound images without knowledge of the patients’ clinical history and pathological results. Any discrepancies were resolved through consultation with a senior radiologist with more than 10 years of experience. The liver nodules were evaluated based on their enhancement features compared to the surrounding normal liver parenchyma. Clearance refers to tumors showing high enhancement in the arterial phase and low enhancement in the portal or delayed phase. Clearance can be classified into three categories: rapid clearance, where tumor enhancement in the arterial phase is significantly lower than that of the surrounding tissue in the portal or delayed phase; slow clearance, where tumor enhancement in the arterial phase is slightly lower than that of the surrounding tissue in the portal or delayed phase; and no clearance, where tumor enhancement in the arterial phase is consistently not lower than that of the surrounding tissue in the portal or delayed phase. The degree of lesion enhancement is further classified as low enhancement, iso-enhancement, or high enhancement. Enhancement types are subdivided into peripheral irregular rim enhancement, diffuse heterogeneous enhancement, and diffuse homogeneous enhancement. The lesion enhancement types are as follows: (1) peripheral irregular rim-like high enhancement, with irregular rim-like high enhancement around the lesion, uneven low enhancement in the center, and strip-like enhancement extending to the lesion center; (2) diffuse heterogeneous hyperenhancement, with both the periphery and center of the lesion showing heterogeneous hyperenhancement; (3) diffuse homogeneous high enhancement, with both the periphery and center of the lesion showing homogeneous high enhancement. Finally, all liver lesions were classified according to the CEUS LI-RADS ( 16 ). Section title: Clinical data and histopathology evaluation Educational score: 4.0788254737854 Domain: biomedical Document type: Study Language: en The preoperative clinical data were collected from medical records, including age, gender, history of hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, liver background (presence or absence of liver cirrhosis), tumor markers: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and carbohydrate antigen 19-9 (CA19-9). Pathological results included hematoxylin-eosin staining and immunohistochemical staining, evaluated by two pathologists with 10 years of work experience, who were blinded to the clinical and radiological information. MVI was defined as the invasion of tumor cells into small blood vessels surrounding the tumor, which can only be detected under a microscope. Patients included in the study were divided into MVI-negative and MVI-positive groups based on pathological findings. Section title: Statistical analysis Educational score: 3.7527832984924316 Domain: biomedical Document type: Study Language: en SPSS 25.0 software (Chicago, IL, USA) was used. Continuous variables with a normal distribution were presented as mean ± standard deviation, and independent samples t-test was used for between-group comparisons. Categorical variables were presented as counts or percentages, and between-group comparisons were conducted using chi-square or rank-sum tests. Multiple-factor logistic regression analysis was employed to identify factors influencing MVI in cHCC-CCA patients. Receiver operating characteristic (ROC) curve was plotted to analyze the predictive value of the influencing factors on MVI. A significance level of P<0.05 was considered statistically significant. Section title: Clinicopathologic characteristics Educational score: 4.0914835929870605 Domain: biomedical Document type: Study Language: en This study included a total of 57 patients, of which 47.4% (27/57) were positive for MVI and 52.6% (30/57) were negative for MVI. The clinical and pathological characteristics of the two groups of patients are compared in Table 1 . There were significant differences between the MVI-positive and MVI-negative groups in terms of tumor size (6.72 ± 3.12 cm vs. 4.29 ± 2.18 cm, p<0.001) and AFP level >400 ng/mL (p=0.046). No significant differences were observed between the two groups in other clinical and pathological data, including age, gender, hepatic background, cirrhosis status, lymph node metastasis, and liver capsule invasion (p>0.05 for all). Section title: CEUS imaging features Educational score: 4.114622116088867 Domain: biomedical Document type: Study Language: en Table 2 summarizes the CEUS features of nodules with opposite MVI statuses. Significant differences were observed in the enhancement pattern and washout degree between the two groups (p = 0.007 and 0.013, respectively) . Among the 27 MVI-positive lesions, 16 (59.3%) exhibited a peripheral nodular enhancement pattern, whereas a similar proportion of MVI-negative nodules (56.7%, 17/30) showed homogeneous enhancement. Regarding washout degree, 59.3% of MVI-positive lesions (16/27) demonstrated pronounced washout within 60 seconds, compared to 26.7% (8/30) in MVI-negative nodules. No significant differences were observed between the two groups in terms of other imaging characteristics (all p > 0.05). According to the CEUS LI-RADS guidelines, 12.3% (7/57) of cHCC-CCA patients were classified as LR-M. However, there was no significant difference in LI-RADS category between the MVI-positive and MVI-negative groups (p = 0.688). Section title: Univariable and multivariable analysis Educational score: 4.097412586212158 Domain: biomedical Document type: Study Language: en According to the results of univariate analysis in Tables 1 , 2 , variables with a p-value < 0.05, including tumor size, AFP > 400 ng/mL, low echo halo, non-smooth tumor contour, enhanced patterns on CEUS, and early washout, were included in the multivariable logistic regression analysis. The results showed that low echo halo (OR = 9.602; 95% CI: 1.009, 91.386; P = 0.049), peripheral irregular rim-like enhancement (OR = 8.360; 95% CI: 1.269, 55.056; P = 0.027), and early washout (OR = 10.041; 95% CI: 1.590, 63.412; P = 0.014) ( Table 3 ) were independent risk factors for MVI in patients with cHCC-CCA (P < 0.05). Subsequently, a receiver operating characteristic (ROC) curve was constructed, and the results showed that the combined diagnostic value was the highest . Section title: Discussion Educational score: 4.131537437438965 Domain: biomedical Document type: Study Language: en MVI, as a pathological criterion, refers to the presence of tumor emboli in the blood vessels of the liver tissue adjacent to the tumor. It is mainly observed in the small branches of the portal vein within the tumor-adjacent tissue, and less frequently in the branches of the hepatic vein, hepatic artery, bile duct, and lymphatic vessels, among others ( 8 , 17 – 19 ). MVI is an important factor affecting the early postoperative recurrence and disease-free survival rate of cHCC-CCA patients. The presence or absence of MVI determines the treatment approach for cHCC-CCA patients. However, currently, MVI can only be detected under a microscope in surgical specimens or biopsy samples from extensively sampled sites, with a significant lag. Therefore, early identification of MVI helps in formulating the optimal treatment strategy, reducing tumor recurrence, and improving prognosis ( 20 – 22 ). CEUS is an emerging imaging technique for tumor microvascular perfusion information. It offers advantages such as non-radiation, real-time operation, and convenience. However, there is limited research on the preoperative prediction of MVI in cHCC-CCA patients using CEUS ( 13 , 23 , 24 ). This study found a certain correlation between ultrasound and CEUS features and MVI in cHCC-CCA patients, demonstrating important preoperative predictive value. Section title: Discussion Educational score: 4.081491470336914 Domain: biomedical Document type: Study Language: en Our research results indicate that the MVI-positive group and MVI-negative group have statistically significant differences in tumor diameter, AFP level, presence of hypoechoic halo around the tumor, irregularity of nodule margins, tumor enhancement pattern, and early washout time. Multivariate logistic regression analysis incorporating the above indicators reveals that the presence of a hypoechoic halo around the tumor, irregular enhancement of nodule margins, and early washout time are independent risk factors for MVI in cHCC-CCA patients. Section title: Discussion Educational score: 4.09066104888916 Domain: biomedical Document type: Study Language: en Univariate analysis in this study confirms that the tumor diameter is larger in the MVI-positive group compared to the MVI-negative group. Some studies have considered tumor size as a prognostic indicator ( 25 , 26 ). It is believed that larger tumors have more surrounding liver tissue and, consequently, increased microvessel density, leading to a higher likelihood of MVI. The presence of irregular tumor margins is usually associated with tumor expansion, protrusion beyond the capsule, and invasion into normal liver parenchyma, reflecting the heterogeneity of tumor cell growth and closely correlating with high invasiveness and poor prognosis. Previous studies have shown that irregular tumor margins have a higher sensitivity in predicting MVI in HCC ( 27 ). Similarly, in this study, the proportion of MVI-positive group with irregular tumor margins was 63.0% (17/27), significantly higher than the 26.7% (8/30) in the MVI-negative group, providing important reference value for the presence of MVI in cHCC-CCA patients. Section title: Discussion Educational score: 4.074705123901367 Domain: biomedical Document type: Study Language: en Serum AFP is an important serological marker for malignant liver tumors. However, there is still controversy regarding its use in predicting MVI before surgery ( 28 – 30 ). In this study, univariate analysis revealed that patients with MVI-positive cHCC-CCA had higher AFP levels. However, multivariate logistic regression analysis did not support AFP as an independent predictor of MVI in cHCC-CCA. This may be attributed to the inherent heterogeneity of tumors and significant inter-individual differences in AFP levels. In clinical practice, 30%-40% of patients with malignant liver tumors still have negative AFP levels even in the advanced stage of the disease. Therefore, further investigation is needed to determine whether AFP can be used for predicting MVI in cHCC-CCA patients, with a larger sample size. Section title: Discussion Educational score: 4.087407112121582 Domain: biomedical Document type: Study Language: en Previous studies have suggested that the continuous outward growth of tumors can compress the surrounding liver tissue and induce fibrotic reactions, which can be visualized as a hypoechoic halo around the tumor on ultrasound ( 31 – 33 ). This hypoechoic halo has been considered as one of the important factors for predicting MVI. However, whether it can serve as an independent high-risk predictor remains controversial, possibly due to the subjective judgment of the operator and the lack of objective criteria. In this study, it was found that the proportion of tumors with a hypoechoic halo around them was significantly higher in the MVI-positive group compared to the MVI-negative group. Both univariate and multivariate analyses demonstrated that the hypoechoic halo around the tumor was an independent risk factor for MVI. Therefore, when predicting the presence of MVI in patients with cHCC-CCA before surgery, the presence of a hypoechoic halo around the tumor is a relatively important indicator. Section title: Discussion Educational score: 4.236652851104736 Domain: biomedical Document type: Study Language: en The degree of tumor differentiation is correlated with rapid washout ( 34 , 35 ). Tumors with low differentiation often exhibit rapid clearance, while those with high differentiation show slow regression. This may be due to (1) the remaining normal hepatic sinusoidal tissue in highly differentiated cHCC-CCA, which causes retention of contrast agents due to the presence of orderly trabecular cells and abundant hepatic sinusoids; (2) the growth of nodules is a progressive process. As the malignancy increases and differentiation decreases, abnormal neovascularization and increased blood supply occur. Normal hepatic artery and portal vein blood supply decrease, resulting in a shortened duration of portal enhancement; (3) the more abnormal neovascularization and arteriovenous shunting in the tumor, the shorter the duration of enhancement and the more pronounced portal clearance. Section title: Discussion Educational score: 4.310332298278809 Domain: biomedical Document type: Study Language: en Washout is defined as the visual decrease in enhancement intensity of liver tumors relative to the surrounding liver background in the arterial phase or thereafter, followed by low enhancement. It has two aspects: washout time and washout degree. The degree of washout is classified as marked or mild washout by comparing the enhancement of the nodule with that of the surrounding parenchyma. Zhu et al. ( 36 ) explored the washout rate of HCC for predicting MVI on contrast-enhanced ultrasound by reviewing imaging data from 271 HCC patients. Their study indicated a significant correlation between early washout and a high likelihood of MVI. Zhou et al. ( 37 ) also found that early washout was an indicator for estimating the occurrence of MVI in HCC in both univariate and multivariate analyses. In contrast, our study found that early washout was an independent risk factor for predicting MVI in cHCC-CCA. The exact mechanism underlying the correlation between washout and MVI remains unclear. This finding may have several explanations. Firstly, tumor microvessel density decreases with the development of MVI, resulting in a reduced dose of contrast agent reaching the tumor site, which leads to attenuation of enhancement and further promotes washout. Secondly, MVI-positive tumors have lower differentiation. Within poorly differentiated tumors, arteriovenous shunting exists, and contrast agents can be completely cleared on contrast-enhanced ultrasound, resulting in a “punched-out” appearance ( 38 , 39 ). Our study found that early tumor clearance time had significant predictive value for MVI in cHCC-CCA patients in both univariate and multivariate analyses. Therefore, we believe that early tumor clearance time, as a simple and intuitive imaging feature during CEUS, is of great value in predicting the presence of MVI in cHCC-CCA patients. ROC curve analysis showed that hypoechoic halo around the tumor, irregular enhancement of the nodule, and early clearance time had an AUC value of 0.8056 for the combined diagnosis of MVI, providing important diagnostic basis for preoperative prediction of MVI in cHCC-CCA patients. Section title: Discussion Educational score: 4.012415885925293 Domain: biomedical Document type: Study Language: en This study has the following limitations: (1) The study was a single-center retrospective study with possible selection bias. (2)The use of contrast-enhanced quantitative analysis software only allows for the selection of a certain portion of the tumor as the research subject when the tumor volume is large, without conducting quantitative analysis on the entire tumor. (3) Subjective bias exists when dividing the tumor morphology into regular or irregular shapes and determining whether the portal phase is rapidly cleared. (4) Ultrasound examinations are prone to interference from intra-abdominal gas and may lead to the omission of isoechoic lesions. Section title: Conclusion Educational score: 3.9971792697906494 Domain: biomedical Document type: Study Language: en There is a certain correlation between the ultrasound and CEUS features and the presence of MVI in patients with cHCC-CCA. Low echo halo around the tumor, peripheral irregular rim-like enhancement of the nodules, and early washout are independent risk factors for MVI in cHCC-CCA patients, which have significant predictive value for the presence of MVI. It provides meaningful reference value for further treatment of patients. However, this study has a relatively small sample size, and multicenter studies are needed to further validate the research findings. | Review | biomedical | en | 0.999996 |
PMC11695214 | Section title: Introduction Educational score: 4.0403594970703125 Domain: biomedical Document type: Review Language: en According to authoritative guidelines such as the National Comprehensive Cancer Network (NCCN), the standard third-line therapy for microsatellite stable (MSS) metastatic colorectal cancer (mCRC) with RAS and BRAF mutations includes regorafenib, fruquintinib, and TAS-102 ( 1 ). The clinical efficacy of standard third-line treatment is limited. Based on the CONCUR ( 2 ), CORRECT ( 3 ), China FRESCO ( 4 ), international RECOURSE ( 5 ), FRESCO-2, and Asia-Pacific TERRA studies ( 6 ), median progression-free survival (mPFS) ranged from 1.9 to 3.71 months, and median overall survival (mOS) ranged from 6.4 to 9.3 months, and these were worse with BRAF V600E mutation, with mPFS of only 1.8 months and mOS of only 4–6 months ( 7 ). More effective treatment options are needed. Section title: Introduction Educational score: 4.0612897872924805 Domain: biomedical Document type: Study Language: en Several small prospective studies ( 7 – 10 ) and retrospective studies have found that tegafur/gimeracil/oteracil (S-1) has shown some efficacy in the third-line treatment of mCRC. Preliminary results from several phase IB/II studies ( 11 , 12 ) of fruquintinib combined with sintilimab for MSS mCRC have demonstrated good efficacy. Theoretically, fruquintinib can restore tumor cell sensitivity to chemotherapy through multi-target and multi-pathway mechanisms. Fruquintinib and chemotherapy can also improve the tumor microenvironment in refractory colorectal cancer patients, promoting the release of tumor-associated neoantigens and enhancing the effect of immune checkpoint inhibitors (ICIs). Section title: Introduction Educational score: 4.072681427001953 Domain: biomedical Document type: Clinical case Language: en The combination of immunotherapy, chemotherapy, and anti-angiogenesis therapy has shown encouraging anti-tumor activity in various refractory solid tumors. However, studies specifically supporting the use of the combination of S-1, fruquintinib, and sintilimab (SFS regimen) in the third-line treatment of MSS mCRC are lacking. The efficacy of the SFS regimen as a third-line treatment for MSS mCRC remains uncertain. Here, we report a case of MSS mCRC with BRAF V600E mutation who achieved a durable response after receiving the SFS regimen third-line treatment. Section title: Case report Educational score: 3.9612083435058594 Domain: clinical Document type: Clinical case Language: en A 23-year-old female patient presented with dizziness in January 2021 and was unable to walk on admission, with an Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) score of 2. She had no significant past medical history or family history. Blood tests showed the following: hemoglobin (Hb), 53g/L; fecal occult blood test positive (3+); carcinoembryonic antigen (CEA), 50.49 ng/L; and carbohydrate antigen 19-9 (CA 19-9), 5,636.00 U/mL. Contrast-enhanced computed tomography (CT) indicated ascending colon cancer and multiple lymph node metastases . A nodule at the S1–S2/3 junction of the liver was suspected to be a liver metastasis . A colonoscopy showed colon cancer. Pathology showed adenocarcinoma of the transverse colon . Immunohistochemistry results were as follows: MSH2, MSH6, MLH1, PMS2 (no loss of expression), CDX-2 (weakly +), CK(−), CK20(+), ER(−), PaX-8(−), and WT-1(−). Genetic testing indicated a BRAF c.1799T>A (p.V600E) missense mutation , with no pathological mutations in KRAS, NRAS, and PIK3CA. No microsatellite instability was detected (MSS type) . Based on these findings, her final diagnosis was adenocarcinoma of the colon with lymph node and liver metastases (cT3N1M1, stage IVc, BRAF-V600E(+), MSS type). Section title: Case report Educational score: 3.8780553340911865 Domain: clinical Document type: Clinical case Language: en After a red blood cell transfusion to improve anemia, the patient received one cycle of FOLFOX chemotherapy (oxaliplatin 85 mg/m 2 + 5-FU 2.4 g/m 2 q2w). Post-chemotherapy, she still experienced significant dizziness and fatigue, with Hb of 66 g/L, CEA of 141.80 ng/L, and CA 19-9 of 8,513.00 U/mL. Figure 3 illustrates the temporal changes in the patient’s CEA and CA 19-9 levels, showing an increase in both markers following the initial treatment. On February 23, 2021, CT showed ascending colon cancer and multiple lymph node metastases similar to previous findings . The metastatic tumor in the S1–S2/3 segment of the liver was slightly enlarged compared to previous findings . The efficacy evaluation was progressive disease (PD). Section title: Case report Educational score: 3.930717945098877 Domain: clinical Document type: Clinical case Language: en The patient failed standardized chemotherapy after one cycle, and severe anemia limited continued chemotherapy. The severe anemia was considered due to continuous bleeding from the colon cancer lesion. Given the patient’s very young age, lack of underlying diseases, a strong desire for treatment, and surgical opinion, she underwent palliative right hemicolectomy + partial gastrectomy + intraoperative microwave ablation of liver tumor (S1 segment metastasis) + adhesiolysis on March 1, 2021. The resected tumor measured 10.5 × 9.0 × 3.0 cm, ulcer-type, with invasion through the intestinal wall . Postoperative pathology showed moderately to focally poorly differentiated adenocarcinoma and partially mucinous adenocarcinoma (approximately 40%) . Immunohistochemistry results were as follows: P53 (70% +), Ki67 (80% +), HER2 (−, score 1 +) , and no loss of expression was observed in MSH2, MSH6, MLH1, and PMS2 . CT indicated that no clear mass was found in the surgical area . The metastatic tumor in the S1–S2/3 segment of the liver was similar to previous findings . A new metastatic tumor in the S6 segment of the liver was found . From April 9, 2021, to August 5, 2021, she underwent seven cycles of FOLFOXIRI (irinotecan 165 mg/m 2 + oxaliplatin 85 mg/m 2 + 5-FU 2.4 g/m 2 q2w) chemotherapy, with bevacizumab (5 mg/kg q2w) added from May 2, 2021, to August 5, 2021. In August 2021, CT showed nodules and masses in the gastric antrum above the anastomosis site, small omental sac, and hepatogastric space, suspected to be metastases . The metastatic tumors in the S1 and S6 segments were similar to previous findings . The efficacy evaluation was PD. Section title: Case report Educational score: 3.8602421283721924 Domain: clinical Document type: Clinical case Language: en From August 2021 to April 2022, the patient received nine cycles of SFS regimen (S-1 60 mg bid po d1-14 + fruquintinib 3 mg qd d1–21 + sintilimab 200 mg ivd q3w). In April 2022, she developed a surgical site ulcer, and CT revealed subcutaneous and intra-abdominal pneumatosis, raising suspicion of an intestinal abdominal wall fistula . The S1 liver metastasis had significantly decreased in size , and the S6 lesion was no longer visible. The patient achieved a partial response (PR), but due to intestinal abdominal wall fistula and abdominal infection, the treatment was suspended. After debridement and infection control, the surgical wound healed well . A repeat CT in July 2022 showed that the subcutaneous pneumatosis at the original upper abdominal incision had been basically absorbed . The S1 segment metastases of the liver were the same as before . From July 26, 2022, to the present, the patient continued to receive an SFS regimen (same as above) for a total of 40 courses. Subsequent regular follow-ups consistently demonstrated stable disease (SD). During this period, the levels of CEA and CA 19-9 exhibited a downward trend and stabilized . Section title: Case report Educational score: 3.4464800357818604 Domain: clinical Document type: Clinical case Language: en The mCRC patient continued to progress after palliative surgery and medical treatment with first-line chemotherapy and second-line targeted combination chemotherapy. The patient achieved a good quality of life due to disease response in the third line of treatment with S-1 combined with fruquintinib and sintilimab. Overall, no serious adverse events were observed. The main adverse reactions were intestinal abdominal wall fistula, mild impairment of liver function, leukopenia, and alopecia. At the time of this report, the patient’s sustained response was ongoing for more than 3 years . Section title: Discussion Educational score: 4.1293535232543945 Domain: biomedical Document type: Study Language: en The standard third-line therapy for MSS mCRC patients is regorafenib, fruquintinib, or TAS-102. However, existing studies indicate that the mOS and mPFS associated with these treatments are generally short, particularly in BRAF V600E-mutated mCRC patients, whose mPFS is only 1.8 months, highlighting the significant challenges of third-line therapy in this patient population. To our knowledge, this is the first report in which the S-1 combined with fruquintinib and sintilimab (SFS) regimen achieved both a durable response and good tolerability as a third-line treatment for MSS mCRC. Notably, this patient experienced a PFS exceeding 3 years following the failure of first- and second-line therapies. Section title: Discussion Educational score: 4.201822280883789 Domain: biomedical Document type: Study Language: en In recent years, ICIs have been introduced in treating MSI-H/dMMR mCRC. However, approximately 85% of mCRC cases are immune-cold MSS tumors, which are unresponsive to ICI therapy. In China, the incidence of MSI-H in colorectal cancer patients is lower (7.7%–10.03%) ( 13 ), further limiting the efficacy of monotherapy with immune checkpoint inhibitors. Anti-vascular endothelial growth factor receptor-tyrosine kinase inhibitors (VEGFR-TKIs) can modulate the tumor immune microenvironment, supporting the theoretical basis for combining anti-VEGFR-TKIs and ICIs in mCRC. Several studies using anti-VEGFR-TKIs and ICIs have shown promising outcomes. The REGONIVO study ( 14 ) first demonstrated that the regorafenib and nivolumab combination regimen was significantly more effective than either agent alone in treating MSS mCRC, achieving an objective response rate (ORR) of 36% and an mPFS of 7.9 months. Similarly, Sun et al. ( 12 ) reported an mPFS of 6.4 months in the fruquintinib plus PD-1 inhibitor (FP) group compared to 3.9 months in the regorafenib plus PD-1 inhibitor (RP) group in refractory MSS mCRC. Preliminary findings from a phase IB/II study of fruquintinib combined with sintilimab for MSS mCRC in China ( 15 ) demonstrated an mPFS of 5.7 months and a mOS of 11.8 months. These findings suggest that fruquintinib plus PD-1 inhibitor may outperform regorafenib plus PD-1 inhibitor, supporting our choice of the fruquintinib and sintilimab combination for this patient. Section title: Discussion Educational score: 4.203076362609863 Domain: biomedical Document type: Study Language: en Third-line chemotherapy alone in mCRC has limited benefit, with a mOS of 6–8 months ( 16 ). Meanwhile, a study by Lin et al. ( 17 ) found that more than half of Asian long-term survivors of mCRC still experience unmet needs, particularly among younger patients (<65 years), who often face financial burdens and limited treatment options during their therapeutic journey. This further underscores the importance of considering a comprehensive approach to treatment strategies in MSS mCRC patients, taking into account their age, financial status, and overall health condition. Anti-VEGFR-TKIs may enhance chemotherapy sensitivity by restoring cellular response mechanisms and overcoming multidrug resistance through multi-target, multi-pathway action, laying a foundation for potential synergy between TKIs and chemotherapy in mCRC. The SUNLIGHT study ( 18 ) compared TAS-102 plus bevacizumab (BEV) to TAS-102 alone in advanced CRC, with mPFS of 5.6 vs. 2.4 months [Hazard Ratio (HR) = 0.44] and mOS of 10.8 vs. 7.2 months (HR = 0.59). The Phase 2 study of Pfeiffer et al. ( 19 ) found that third-line BEV + TAS-102 significantly reduced the risk of death in BEV-naïve patients compared to those with prior BEV treatment, with HRs of 0.39 vs. 0.76. However, the retrospective study of Bang YH et al. ( 20 ) indicated that in patients who had previously been treated with BEV, the mOS with BEV combined with capecitabine was significantly worse compared to that of patients who had not received BEV treatment (p = 0.018). The retrospective study of Bang YH et al. ( 20 ) indicated that BEV combined with capecitabine significantly worsened mOS in patients with prior BEV exposure (p = 0.018). Hence, re-challenging BEV + chemotherapy as third-line treatment remains limited, and we did not choose the BEV + TAS-102 regimen for this patient. Section title: Discussion Educational score: 4.12666130065918 Domain: biomedical Document type: Study Language: en In the treatment of CRC liver metastasis, a study by Sawano et al. ( 21 ) suggests that adjuvant chemotherapy should be considered for CRC patients following liver metastasis resection to reduce the risk of hepatic recurrence. Previous meta-analyses have demonstrated that, in the treatment of metastatic colorectal cancer, S-1 therapy offers comparable efficacy in PFS, ORR, and OS compared to those of 5-FU-based regimens, with a reduced toxicity profile ( 22 ). These findings support the use of S-1 in metastatic colorectal cancer patients who are intolerant to 5-FU-based therapies. Lee et al. ( 11 ) conducted a single-arm phase II study of 19 patients with mCRC who had failed standard oxaliplatin or irinotecan chemotherapy and were treated with S-1 with a median time to progression (TTP) of 2.1 months and a mOS of 11.3 months. This suggests that S-1, as a third-line treatment for patients with mCRC, can prolong the patient’s life and is safe and effective. For anti-VEGFR-TKI combined with S-1, Li et al. ( 23 ) conducted a single-arm phase II study to investigate S-1 combined with apatinib in the treatment of mCRC and included 30 patients with mCRC who had previously received more than two standard chemotherapies (irinotecan and oxaliplatin). The results showed that the mPFS and mOS were 7.9 months and 12.9 months, respectively. These results suggest that S-1 combined with anti-VEGFR-TKI may be used as a therapeutic exploration mode for the treatment of mCRC in the future, so we tentatively selected S-1 for the chemotherapeutic drugs of this patient. Section title: Discussion Educational score: 4.191692352294922 Domain: biomedical Document type: Study Language: en The BRAF V600E mutation, linked with aggressive disease and poor response to chemotherapy, presents a significant clinical challenge. The optimal regimen for these cases—FOLFOXIRI combined with BEV post-palliative surgery—offers limited benefit, with PFS of approximately 4 months. Standard chemotherapy in BRAF V600E-mutated mCRC yields an ORR below 10%, mPFS of ~1.8 months, and mOS of 4–6 months ( 7 ). Vemurafenib, a BRAF V600E inhibitor, has shown efficacy in melanoma and lung cancer, but not in mCRC. The BEACON study ( 24 ) showed that combining encorafenib, binimetinib, and cetuximab achieved an mPFS of 8 months and mOS of 15.3 months, yet the lack of approval and high costs in China made this approach unfeasible for our patient. Section title: Discussion Educational score: 4.0941877365112305 Domain: biomedical Document type: Study Language: en Combining anti-VEGFR-TKIs, ICIs, and chemotherapy may enhance immune recognition and T-cell reactivity through immune microenvironment modulation, synergizing the efficacy of immune therapies ( 25 ). This combined strategy has been effective and safe in treating several refractory solid tumors, such as non-small cell lung cancer post-EGFR-TKI resistance, extensive-stage small cell lung cancer, and advanced cervical cancer [e.g., IMpower151, ORIENT31 ( 26 ), ETER701 ( 27 ), HARMONi-A ( 28 ), and BEATcc ( 29 )]. Therefore, these results provide a basis for our patients receiving chemotherapy combined with anti-VEGFR-TKI and PD-1/PD-L1 combination mode. In our report, this patient sustained remission for more than 3 years. Therefore, it is promising to explore chemotherapy combined with ICIs and anti-VEGFR-TKI as a third-line treatment for mCRC. Section title: Discussion Educational score: 3.035245180130005 Domain: clinical Document type: Clinical case Language: en Finally, the patient tolerated the adverse reactions well except for intestinal abdominal wall fistula and abdominal infection. However, we did not consider these adverse events to be related to this third-line combination regimen, as the patient received palliative surgery followed by continuous BEV treatment, considering the adverse reactions of BEV resulting in infection and abdominal wall fistula caused by poor wound healing. We stopped the drug for 3 months and then continued to use this regimen for nearly 3 years without related adverse reactions, and the fistula healed well. Section title: Conclusion Educational score: 3.757774591445923 Domain: biomedical Document type: Clinical case Language: en In this report, we describe a patient with MSS mCRC with BRAF V600E mutation who showed a durable response to third-line treatment with an SFS regimen. This strategy of SFS may provide effective third-line treatment options for patients with mCRC. Therefore, it is necessary to evaluate in future clinical trials . | Review | biomedical | en | 0.999996 |
PMC11695215 | Section title: Introduction Educational score: 4.215209007263184 Domain: biomedical Document type: Review Language: en Recurrent spontaneous abortion (RSA), also referred to as recurrent pregnancy loss (RPL), is a significant concern within women’s reproductive health. This condition is linked to both subsequent obstetric complications and enduring health issues, such as cardiovascular disease and mental health implications ( 1 ).It is estimated that RSA impacts approximately 1% to 5% of couples attempting to conceive ( 2 ). The etiology of RSA is complex and has obvious heterogeneity ( 3 ). Numerous risk factors have been identified in association with RSA, including chromosomal abnormalities, infections, endocrinopathies, uterine anomalies, antiphospholipid syndrome (APS), inherited thrombophilia and lifestyle factors ( 4 ). Additionally, factors such as improper decidualization ( 5 ), and the presence of autoantibodies have also been implicated in RSA ( 6 ). Nevertheless, approximately 40-50% of RSA cases are classified as URSA, where the cause of miscarriage remains unknown ( 7 ). Furthermore, the lack of consensus regarding the definition of RSA, specifically whether it should be characterized by two or more versus three or more pregnancy losses, complicates the prevention and management of this condition ( 8 ). Section title: Introduction Educational score: 4.0001325607299805 Domain: biomedical Document type: Review Language: en Various therapeutic interventions have been utilized in the management of RSA in recent decades ( 9 – 14 ). Key treatments are widely recognized to encompass progesterone supplementation, levothyroxine for individuals with hypothyroidism and RSA, and the concurrent administration of heparin and aspirin for patients diagnosed with APS ( 15 ). It is important to note that anticoagulant therapy is generally not advised for women with hereditary thrombophilia and RSA ( 16 ), except when necessary for the prevention of venous thromboembolism (VTE). However, the existing evidence is inconclusive regarding the efficacy of certain immunomodulatory agents, such as intravenous immunoglobulin (IVIG), intralipid, and granulocyte colony-stimulating factor (G-CSF), in the treatment of URSA. While some studies have reported an improvement in live birth rates (LBR) among patients with URSA following the administration of immunomodulatory agents, others have failed to demonstrate a significant difference ( 17 , 18 ).Therefore, further high-quality clinical research is imperative to enhance clinical practice and inform strategies for the treatment and prevention of RSA ( 15 ). Section title: Introduction Educational score: 3.97320294380188 Domain: biomedical Document type: Other Language: en Clinical studies are the cornerstone of evidence-based medicine, with quality and scope being crucial. The establishment of the ClinicalTrials.gov database was designed to improve transparency and reduce publication bias, ensuring the integrity of study outcomes ( 19 ).Currently,ClinicalTrials.gov serves as the largest and most thorough repository of data on active and completed clinical research worldwide ( 20 , 21 ), featuring registration details for roughly 500,000 studies conducted in more than 200 nations. This registry encompasses a wide array of research pertaining to pharmaceuticals, therapeutic interventions, medical devices, and behavioral strategies, providing comprehensive trial data including objectives, eligibility criteria, design, status, locations, and outcomes ( 22 ).The extensive information available on ClinicalTrials.gov offers a distinctive opportunity to comprehend the landscape of clinical trials for specific disease groups or indications. Section title: Introduction Educational score: 3.8784420490264893 Domain: biomedical Document type: Review Language: en RSA poses a considerable challenge within the field of reproductive medicine ( 23 ). Despite extensive efforts to elucidate its underlying etiologies, effective treatment options remain limited ( 24 ). RSA is attracting increasing scholarly attention. A recent Lancet Series published in 2021 critically assessed the existing evidence on miscarriage ( 15 , 25 , 26 ), and an increasing number of clinical studies have been undertaken to investigate the scientific basis of RSA. Consequently, a comprehensive understanding of the current characteristics of RSA clinical trials is essential for improving trial design and identifying neglected areas of research. Section title: Introduction Educational score: 3.0292270183563232 Domain: biomedical Document type: Study Language: en Prior research has investigated the attributes of clinical trials listed in the Clinical Trials.gov database across various medical conditions ( 27 – 31 ).However, there is no study focusing on the landscape of RSA clinical trials. The aim of this study is to characterize the various aspects of these trials, such as study design, enrollment, location, and study population in order to provide valuable insights for policy makers, the medical research community and pharmaceutical companies. Section title: Materials and methods Educational score: 4.056557655334473 Domain: biomedical Document type: Study Language: en As of March 2,2024, a total of 485,171 clinical trials were registered on Clinical Trials.gov. A search was conducted on the Clinical Trials.gov website in March 2024 using the keywords recurrent pregnancy loss, recurrent miscarriage, recurrent abortion, and habitual abortion. Subsequently, the four arms of the identified trials were combined and any duplicates were eliminated. A manual assessment of the study conditions and titles of these trials was carried out to exclude those not relevant to RSA. Ultimately, a total of 138 clinical trials met the inclusion criteria for this study, as depicted in Figure 1 . Section title: Materials and methods Educational score: 2.6265904903411865 Domain: biomedical Document type: Study Language: en The data collected for analysis included the study title, study type, number of sites, enrollment, funder type, status, start date, locations, condition, trial phases, design, participant gender, and intervention type. A manual review was conducted for study population and number of miscarriages. Study population was defined as individuals eligible for participation in a study. The study was deemed exempt from institutional review board oversight as it solely utilized publicly accessible data devoid of personal identifiers or involvement of human subjects. Section title: Statistical analysis Educational score: 2.0722415447235107 Domain: biomedical Document type: Study Language: en Descriptive analyses indicate that categorical variables were represented using both numerical counts and percentages. Data processing and analysis were conducted using Microsoft Office Excel. Section title: Basic characteristics of registered RSA trials Educational score: 4.135078430175781 Domain: biomedical Document type: Study Language: en A total of 138 clinical studies were identified as being related to RSA, consisting of 72 interventional studies (52.17%) and 66 observational studies(47.83%). The majority of trials began in 2010 or later (121,87.68%). The progression of the number of trials over time is illustrated in Figure 2 . The majority of trials were conducted at single-center facilities (108,78.26%). Of the 138 studies, nearly half had a planned or actual enrollment of 100 patients or fewer(67,48.55%), while only 15 (10.87%) studies enrolled more than 500 patients. The majority of studies included in the analysis were funded by sources categorized as “other” (130,94.20%), with a small percentage funded by industry (6, 4.35%) and NIH/US Federal funding (2,1.45%). Among the 138 studies related to RSA, 60 (43.48%) were completed, 35 (25.36%) had an unknown status, and 15 (10.87%) were actively recruiting participants. The majority of trials included only female participants (130,94.20%). Table 1 provides an overview of basic clinical trial characteristics. Section title: Geographical distribution and transnational collaborative trials Educational score: 3.8951852321624756 Domain: biomedical Document type: Study Language: en Among the clinical trials examined, 14 did not specify their respective regions. The remaining 124 trials were conducted across various continents. Figure 3 illustrates the geographical distribution and transnational collaborative relationships of these trials. In this figure, each node represents an individual country, with its color indicating the continent to which the country belongs. The size of the node correlates with the number of trials conducted by that country. Notably, Asia hosted the highest number of clinical trials (46,33.33%), followed by Europe (36,26.09%), Africa (29,21.01%), America (13,9.42%). China hosted nearly one-fifth of all clinical trials (29,21.01%), within three trials in Hong Kong and two in Taiwan. This was followed by Egypt (28,20.29%), Denmark (8,5.80%), Turkey (8,5.80%), and the United States (8,5.80%). Section title: Geographical distribution and transnational collaborative trials Educational score: 2.1753878593444824 Domain: biomedical Document type: Other Language: en The straight line symbolized the cooperative relationship between countries, indicating that the nodes at both ends of the line corresponded to two countries engaged in international clinical trials. Conversely, nodes not connected to others by a straight line, but instead linked by a curved line pointing to themselves, signified that the country conducted only domestic, rather than transnational, clinical trials. Consequently, European countries demonstrated a transnational collaborative relationship in trials, with the United Kingdom conducting trials with Spain, and Austria collaborating with Poland and Germany. Section title: Study population Educational score: 4.093278408050537 Domain: biomedical Document type: Study Language: en Figure 4 illustrates the distribution of clinical trials based on the study population. The majority of trials (61,44.20%) focused on individuals with URSA. Additionally, a considerable proportion of patients with RSA (47,34.06%) were included in trials without specific categorization based on the underlying etiology. Other prevalent study populations encompassed thrombophilia (14,10.14%), thyroid disease (4,2.90%), immunological factors (3,2.17%), male factors (2,1.45%), and insulin resistance (2,1.45%). Out of the 138 studies related to RSA,75 (54.35%) clinical trials involved women who experienced two or more pregnancy losses, while 38 (27.54%) clinical trials included women with three or more losses. Only 50 (36.23%) clinical trials explicitly stated that the miscarriages were consecutive. Section title: Characteristics of interventional studies Educational score: 4.06589412689209 Domain: biomedical Document type: Study Language: en A total of 72 interventional studies were included in the analysis, with their characteristics detailed in Table 2 . The majority of these studies (60,83.33%) utilized a parallel assignment, while a smaller percentage (11,15.28%) employed a single group assignment. Only one study utilized a factorial study design. In terms of allocation strategy, the majority of studies (53, 73.61%) employed a randomized approach, while a smaller percentage (10, 13.89%) used a non-randomized method. Additionally, a further 9 studies (12.50%) did not provide information on the allocation strategy. All interventional trials included in the analysis reported information on masking, with 35 trials (48.61%) utilizing blinding techniques. Section title: Characteristics of interventional studies Educational score: 4.128499507904053 Domain: biomedical Document type: Study Language: en A total of 78 interventions were identified in various clinical trials. These interventions were categorized as follows: drug (49,62.82%), behavioral interventions (7,8.97%), procedures (7,8.97%), genetic interventions (4,5.13%), devices (3,3.85%), biological interventions (2,2.56%), dietary supplements (2,2.56%), diagnostic interventions (1,1.28%), and other interventions (3,3.85%). Figure 5 provides a comprehensive breakdown of each intervention type. Within the drug category, anticoagulants were utilized in 12 studies (24.49%), and immunotherapy was employed in 21 studies (42.86%). Figure 6 presents a pie chart illustrating the distribution of study drugs in registered interventional studies. Notably, approximately one-third of these drug intervention trials did not specify phase data, while slightly more than half were classified as Phases 2-3 or 4 (51.39%). Figure 7 depicts the number of trials conducted at each phase across various types of drug interventions. Additionally, there is a notable increase in the intervention types from 2010 to 2024, with a particular rise in behavioral intervention trials. Figure 8 presents the annual number of RSA clinical trials categorized by intervention type. Section title: Discussion Educational score: 3.1517207622528076 Domain: biomedical Document type: Study Language: en The objective of this study was to delineate the landscape of registered RSA clinical trials on Clinical Trials.gov, with the intention of establishing a foundation for the advancement of disease prevention and treatment within the realm of RSA. Section title: Discussion Educational score: 3.8615059852600098 Domain: biomedical Document type: Study Language: en Our study revealed that RSA related clinical trials comprised a mere 0.03% of all registered trials. The majority of these trials were of a small scale and received funding primarily from sources other than industry or NIH. The limited number of high-quality clinical trials in this area may be attributed to the historically low priority given to miscarriage ( 32 ).However, it is crucial to emphasize the necessity for high-quality research in this domain, given the association of RSA with enduring health issues, financial strains, and psychological anguish ( 15 , 25 , 26 , 32 ). Section title: Discussion Educational score: 4.058711528778076 Domain: biomedical Document type: Study Language: en The findings of previous meta-analyses did not indicate significant geographical variations in the prevalence of RSA ( 33 ). However, the distribution of RSA related clinical trials did not align consistently with regional incidence rates of RSA. Our study revealed that Asia conducted the highest number of registered trials, with China and Egypt being the leading countries in terms of trial quantity. Additionally, Turkey, the United States, and Denmark each hosted eight clinical trials, placing them jointly in the third position. This disparity in trial distribution may be explained by societal and cultural factors that contribute to the stigmatization, discrimination, and ostracism faced by childless women, particularly in low-income regions ( 15 ). Furthermore, there is a rising prevalence of RSA in both the United States ( 34 ) and China ( 35 ). This escalating incidence of RSA in these nations may have led to an increased emphasis on conducting clinical trials in this area. Section title: Discussion Educational score: 3.9935264587402344 Domain: biomedical Document type: Study Language: en Men were significantly underrepresented in clinical trials pertaining to RSA, with only 5.80% of such trials including male participants. Despite the fact that half of the embryo’s genetic material is contributed by the male partner, research has predominantly focused on maternal factors such as gene mutations, endocrine disorders, and uterine anatomical abnormalities. Recent studies have suggested that male factors may also be influential in the occurrence of RSA ( 36 – 40 ). One retrospective study has have reported that sperm DNA fragmentation is markedly higher in men experiencing RSA compared to fertile controls ( 41 ). However, A prospective cohort study suggest that sperm DNA fragmentation does not appear to be associated with RSA ( 42 ), highlighting the necessity for further investigation in this domain ( 43 ). Section title: Discussion Educational score: 4.135609149932861 Domain: biomedical Document type: Study Language: en Our analysis revealed that the URSA population has been the focus of the majority of studies, as URSA accounts for nearly half of all RSA cases ( 7 ).There is a notable concern regarding the inclusion of women with a history of pregnancy losses in clinical trials, with 75 (54.35%) trials specifically enrolling women after two or more losses and 38 (27.54%) trials enrolling women after three or more losses. A significant portion of the studies (50,36.23%) reported that the miscarriages were consecutive. Prioritizing the accurate diagnosis of RSA is crucial in addressing the heightened data heterogeneity arising from variations in study populations, which may be attributed to a lack of consensus on the definition of RSA ( 44 – 47 ).For instance, the Royal College of Obstetricians and Gynecologists (RCOG) defines RSA as a minimum of three first-trimester miscarriages ( 43 ),while the European Society of Human Reproduction and Embryology (ESHRE) ( 3 )and Chinese guidelines define it as at least two miscarriages ( 48 ).Additionally, only the Chinese guidelines incorporate consecutive miscarriages. Establishing a uniform definition is crucial not only for standardizing protocols, but also for promoting consistency in research and clinical practice ( 46 ). Section title: Discussion Educational score: 4.168792724609375 Domain: biomedical Document type: Study Language: en The results of our study suggest that nearly half of clinical trials focusing on RSA are interventional studies. Clinical trials has primarily focused on drug interventions, particularly anticoagulants, which are commonly utilized as a preventive measure for women with APS and are increasingly administered to women with URSA ( 49 ).However, ESHRE advise against the use of heparin for women with URSA ( 3 ).This recommendation is supported by evidence from two multi-center randomized trials, which found no significant benefit of heparin compared to placebo or multivitamin supplementation ( 50 , 51 ).Additionally, several trials have investigated the efficacy of new medications, such as IVIG, intralipid, and G-CSF, in addressing the clinical needs for URSA treatment. A high-quality randomized controlled trial (RCT) identified that IVIG administration significantly enhances LBR in women experiencing four or more unexplained pregnancy losses ( 52 ). Based on these findings, ESHRE conditionally recommends the early administration of repeated high-dose IVIG therapy in pregnant women with URSA ( 3 ). However, the efficacy of G-CSF remains inconclusive, as evidenced by conflicting outcomes from two clinical trials involving women with URSA. In one RCT, G-CSF was associated with an improved LBR compared with placebo ( 53 ). Conversely, in a larger RCT, G-CSF did not demonstrate any benefit ( 54 ).Given the high quality of the latter trial, ESHRE considers its findings to supersede earlier reports and does not recommend the use of G-CSF in patients with URSA ( 3 ). In addition, ESHRE does not recommend intralipid therapy for URSA due to insufficient evidence ( 3 ). Section title: Discussion Educational score: 3.7051310539245605 Domain: biomedical Document type: Review Language: en While clinical studies on RSA drugs constitute the majority of intervention research, novel treatment approaches, particularly behavioral interventions, are continually being developed. At present, seven pertinent studies are registered on ClinicalTrials.gov, with three having reached completion , however, their findings have yet to be disclosed. Notably, some research has indicated a correlation between stress, obesity, and RSA ( 25 , 55 – 60 ).Both ESHRE and RCOG advocate for additional research to advance psychological support and lifestyle modifications for RSA women and their partners ( 3 , 43 ). Section title: Discussion Educational score: 3.9442360401153564 Domain: biomedical Document type: Study Language: en Our study has some limitations. Firstly, it should be noted that Clinical Trials.gov does not encompass all clinical trials related to RSA conducted globally, as a portion of trials are registered in alternative databases such as the Chinese clinical trial registry or European union clinical trial Registry. Although we considered incorporating additional registries into our analysis, doing so could potentially compromise the level of detail and accuracy afforded by utilizing a single data source. The data fields across different registries are not uniform, either in terms of their existence or their coding, and the reporting standards of some international registries may not be applicable to ClinicalTrials.gov ( 61 ). Secondly, certain trials may have omitted crucial elements or keywords during registration. Lastly, misclassification may potentially result in erroneous conclusions being drawn from the data. Section title: Conclusion Educational score: 3.650355577468872 Domain: biomedical Document type: Review Language: en This research offers a comprehensive overview of global clinical trials related to RSA. Our results indicate that the research activity of RSA is inadequate for the progression of prevention and treatment strategies. There is a pressing need for high-quality research and further investigation into the roles of psychological support and male factors in RSA. | Review | biomedical | en | 0.999997 |
PMC11695220 | Section title: Introduction Educational score: 3.996086359024048 Domain: biomedical Document type: Review Language: en Kaposi’s sarcoma (KS) is a pigmented, angiosarcoma-like soft-tissue lesion that is commonly associated with human herpes virus 8 (HHV-8) infection. KS typically affects the skin, mucous membranes, lymphatic system, and gastrointestinal tract, and is most frequently observed in patients with compromised immune systems. The four main subtypes of KS are classical, African, transplantation, and AIDS-related. Section title: Introduction Educational score: 3.9329278469085693 Domain: biomedical Document type: Review Language: en KS is a common malignancy in patients with AIDS and is classified as an AIDS-related malignancy ( 1 ), also known as AIDS-KS, commonly arises in the advanced stages of HIV infection in affected individuals ( 2 ). The incidence of KS is 20,000 times higher among HIV patients who do not receive highly active antiretroviral therapy (HAART) ( 3 ). AIDS-KS typically affects the upper body and can cause damage to internal organs in addition to the skin. The disease progresses rapidly, is difficult to treat, and has a high mortality rate. Section title: Introduction Educational score: 3.91847825050354 Domain: biomedical Document type: Clinical case Language: en In recent years, there has been an increasing amount of research on the use of PD-1 inhibitors for treating various malignant tumors. However, there is limited data available on their effectiveness in treating KS. A case report describes an 82-year-old man with metastatic KS who achieved complete remission after 12 sessions of treatment with navulizumab, despite not being HIV-positive ( 4 ). In a phase II clinical trial studying the PD-1 inhibitor pembrolizumab for treating classic KS, 2 patients achieved complete remission, 10 had partial remissions, and 5 had stable disease after 204 months of follow-up. These results demonstrate that the PD-1 inhibitor has better therapeutic efficacy in patients with KS ( 5 ).but the efficacy of PD-1 inhibitors in the treatment of the AIDS-KS population is not clear. Here, we present a patient with AIDS-KS who achieved complete remission with the application of tirilizumab. Section title: Case presentation Educational score: 3.763369560241699 Domain: clinical Document type: Clinical case Language: en A male patient, aged 30, presented with a facial rash that had been gradually increasing. He had been diagnosed with eczema and infectious dermatitis in several dermatology departments and had been treated with fusidic acid cream and loratadine tablets, but with no improvement. The rash then worsened, spreading to the neck and upper limbs. The patient presented with fever, tightness in the chest, and shortness of breath after activity. He was admitted to our hospital on 31st December 2022 and diagnosed with AIDS. Blood cultures were positive for Talaromyces marneffei, and cervical lymph node aspiration biopsy was consistent with lymph node tuberculosis. The blood test for tolulized red unheated serum test (TRUST)was 1:16. During hospitalization, a purplish-red mass measuring approximately 2cm x 3cm with a hard texture grew on the right sole of the foot. A surgical-assisted mass excision was performed, and biopsy pathology confirmed it to be consistent with KS . Section title: Case presentation Educational score: 3.8848445415496826 Domain: clinical Document type: Clinical case Language: en The patient’s CD4+ T-lymphocyte count was initially only 3 cells/μL, and their HIV-RNA count was 3.26×10 5 copies/ml. They received highly effective antiretroviral therapy(HARRT)with tenofovir + lamivudine + dolutegravir, amphotericin B for t Talaromyces marneffei infections, isoniazid, moxifloxacin, and rifabutin for anti-lymphatic node tuberculosis, and benzylpenicillin for syphilis. After two months of hospitalization, the patient’s CD4+ T-lymphocyte count increased to 23 cells/μL, HIV-RNA was undetectable, the generalized rash subsided and disappeared, and the uncomfortable symptoms such as fever, chest tightness, and shortness of breath improved significantly, enabling the patient to carry out daily activities. For the treatment of KS, doxorubicin liposome was administered at a dose of 40mg every three weeks. After six consecutive applications, the purplish-red mass on the sole of the foot disappeared. The treatment course ended on 26th April 2023 and the patient achieved a significant improvement. Section title: Case presentation Educational score: 3.9158084392547607 Domain: clinical Document type: Clinical case Language: en After one month of follow-up, the patient did not experience a recurrence of the generalized rash and fever. However, on 26th May 2023, the patient returned to the clinic with a purplish-red macular rash on the left thigh root and both calves. The rash was accompanied by a hard nodule on the thigh root and thickening of the leg circumference. Additionally, the patient experienced obvious swelling in both calves after activity. A skin biopsy was performed on the thigh maculopapular rash, and the biopsy’s pathological evaluation was consistent with KS, the immunohistochemical results were as follows: CD34 (-), D2-40 (+), ERG (+), HHV8 (+), Ki-67 (80%+), S-100 (-), Vimentin (+). The diagnosis was a recurrence of KS, and doxorubicin liposome 40 mg/dose was administered again, once every three weeks. After five consecutive applications, there were no obvious changes in the patient’s lower limb maculopapular rash. At present, the CD4+ T-lymphocyte count was 51 cells/μL and HIV-RNA was below the lower limit of detection. Section title: Case presentation Educational score: 4.015778064727783 Domain: clinical Document type: Clinical case Language: en After obtaining informed consent from the patient, tirilizumab 200 mg/dose was added to the original regimen on September 19, 2023. The medication was administered once every three weeks. After nine consecutive uses, the patchy rash at the root of the left thigh reduced in range, the hard nodules disappeared, and the color became significantly lighter . Additionally, there was no swelling in both calves, indicating a promising therapeutic effect. Despite the absence of symptoms indicative of thyroid dysfunction, namely weakness, fatigue, facial swelling, or muscle pain, thyroid function tests revealed an elevated thyrotropin (TSH) level of 16.19 μIU/mL, a triiodothyronine (T3) level of 1.51 nmol/L, and a total thyroxine (T4) level of 107.09 nmol/L. The endocrinologists diagnosed PD-1-induced hypothyroidism and prescribed 25 μg levothyroxine tablets for treatment. To assess the therapeutic effect, skin biopsies were performed on the macules on the root of the thighs and on the calf of the left lower limb, respectively. Pathological assessment revealed chronic vasculitis . and the patient achieved complete remission. The timeline of patient diagnosis and treatment is shown in Figure 4 . Section title: Discussion Educational score: 4.128036975860596 Domain: biomedical Document type: Review Language: en The optimal treatment of AIDS-KS is yet to be standardized. In cases of only local skin lesions, alitretinoin, vincristine and bleomycin are all used for local treatment, and skin lesions are highly sensitive to radiotherapy, with complete remissions observed in about 95% of cases ( 6 ). However, local treatment is rarely used because it has no effect on systemic disease and radiation therapy may cause long-term local toxicity. Timely and effective highly active antiretroviral therapy (HAART) can significantly reduce the incidence of AIDS-KS ( 7 ), but some patients are unable to avoid AIDS-KS despite HIV viral load suppression and impressive T-lymphocyte counts ( 8 ), and HIV may be directly involved in carcinogenesis early in the human body. HAART combined with systemic chemotherapy is the current preferred treatment approach for AIDS-KS, with liposomal anthracyclines being the favored option. The combined use of HAART and liposomal anthracycline chemotherapy in advanced KS achieves a 70% overall remission rate and prolonged remission. However, it is unclear whether the treatment provides long-term benefits, and prolonged regimens increase the risk of adverse events, which limits the treatment of relapsed patients ( 9 ). Section title: Discussion Educational score: 4.524094581604004 Domain: biomedical Document type: Study Language: en In recent years, immunotherapy has become a popular research topic for various diseases. The recombinant programmed cell death protein 1/ligand L1 (PD⁃1/PD⁃L1) pathway is capable of hindering the effector phase of cancer-specific immune responses. Several studies have identified that PD-L1 expression intensifies during viral lysis and replication after Kaposi’s sarcoma-associated herpesvirus(KSHV) infects human mononuclear cells, indicating a novel immune evasion strategy for KSHV ( 10 ). The expression of HHV-8 viral antigen results in increased expression of PD-1 and PD-L1 on the surface of antigen-specific T cells. This, in turn, leads to higher response rates to anti-PD-1 and anti-PD-L1 drugs ( 11 ). It has been discovered that PD-1 expression in natural killer cells of KS patients is linked to functional natural killer cell exhaustion. This, in turn, promotes the immune escape of tumor cells ( 12 ). Anti-PD-1 or anti-PD-L1 antibodies can block the interaction between PD-1 and PD-L1, which lifts the inhibition of T-lymphocyte proliferation. Section title: Discussion Educational score: 4.110940456390381 Domain: biomedical Document type: Study Language: en This reversal of the tumor immune microenvironment reactivates the ability of T-lymphocyte to lyse cancer cells. PD-1 inhibitors promote the proliferation of CD4+ T lymphocytes, which is consistent with the clinically observed changes in CD4+ T lymphocytes . It is speculated that improved HIV immune reconstitution promotes immune-mediated enhanced control and clearance of HHV-8, providing another possible avenue for AIDS-KS treatment. Section title: Discussion Educational score: 4.061850070953369 Domain: biomedical Document type: Clinical case Language: en The use of PD-1/PD-L1 inhibitors is theoretically more appropriate for patients with HIV-associated cancers, but in practice the unknown adverse effects that may be caused by the immunocompromised state of HIV patients have limited their use in this population. In this case, the patient had multiple opportunistic infections and had been taking multiple medications orally for an extended period. repeated application of PD-1 inhibitors and chemotherapy resulted in a gradual increase in the number of CD4+ T-lymphocyte, and a complete remission was achieved for relapsed and refractory AIDS-KS. Only mild malaise and fatigue were observed throughout the course of treatment, along with elevated thyroid-stimulating hormone. No other grade 2 or higher immune-related adverse reactions occurred. In addition to good efficacy, the encouraging results were attributed to fewer side effects and better tolerability. Section title: Discussion Educational score: 4.400446891784668 Domain: biomedical Document type: Review Language: en In patients with AIDS-KS, vigilance is required for Kaposi Inflammatory Cytokine Syndrome (KICS), a serious disease state associated with KS that has only been proposed in recent years. KICS is characterized by a systemic inflammatory response and excessive release of multiple inflammatory cytokines. Clinical manifestations include skin lesions such as purplish-red or dark blue plaques, nodules or tumors, which may Systemic symptoms may also manifest, including fever, fatigue, and weight loss. Additionally, lymph node enlargement and organ dysfunction may occur. KICS is prone to occur in AIDS patients with a lower CD4+ cell counts, often accompanied by HHV-8 infections. The inflammatory milieu of KICS may facilitate the progression of Kaposi’s sarcoma ( 13 ). Additionally, both KICS and immune reconstitution syndrome (IRIS) are associated with human immunodeficiency virus (HIV) infection and an abnormal immune system response. These two conditions may co-occur in HIV-infected individuals and may also influence each other. The recovery of the immune system may intensify the reactivity of the inflammatory response, resulting in an exacerbation of KICS symptoms. Conversely, the inflammatory response associated with KICS may influence the development of IRIS, thereby complicating the recovery of the immune system. With a clinical mortality rate of up to 60% ( 14 ), KICS requires more accurate identification by clinicians to prevent misdiagnosis and to enhance clinical outcomes. Section title: Conclusion Educational score: 3.789889097213745 Domain: biomedical Document type: Study Language: en Due to the complex pathogenesis of AIDS-KS, outdated basic research and the lack of specific therapeutic approaches, the treatment of AIDS-KS continues to be a challenge. The PD-1 inhibitor may enhance the proliferation of CD4+ T lymphocytes and improve immune reconstitution. This could potentially serve as a novel approach for successful treatment. This treatment shows promise due to its efficacy, few adverse effects and good tolerability. However, this study is only a case report of a clinical treatment, which has significant limitations and requires further exploration and research. | Review | biomedical | en | 0.999997 |
PMC11695221 | Section title: Introduction Educational score: 3.0175769329071045 Domain: biomedical Document type: Other Language: en Breast cancer is a significant global health concern, recently surpassing lung cancer as the most commonly diagnosed cancer worldwide ( 1 ). It poses a severe threat to the health of women and remains a critical public health challenge. The current gold standard for diagnosing breast cancer involves invasive procedures, which carry the risk of complications and can damage healthy tissues ( 2 ). Section title: Introduction Educational score: 4.275447845458984 Domain: biomedical Document type: Study Language: en Conventional ultrasound imaging is a widely used method for diagnosing breast masses ( 3 , 4 ), However, its ability to distinguish between benign and malignant masses is limited ( 5 ). B-mode ultrasound can provide information about the size, shape, boundary, and internal characteristics of breast masses but is insufficient for detecting blood flow within these masses ( 6 ). To supplement B-mode ultrasound, color Doppler flow imaging (CDFI) is used to visualize blood flow distribution within breast masses. However, the overlapping imaging features of benign and malignant masses often necessitate further follow-up and biopsies. CDFI can only show the diameter of >0.2mm vessels and relatively high blood flow rates (>1cm/s) ( 7 , 8 ). Due to the physical diffraction limit, the spatial resolution of conventional ultrasound imaging, including contrast-enhanced ultrasound (CEUS), which is widely used in clinical practice, is limited by the ultrasound wavelength, and cannot distinguish targets smaller than half a wavelength ( 8 – 11 ). Although it is possible to reduce the wavelength and thus improve the spatial resolution by increasing the ultrasound frequency, the increase in attenuation inevitably induces a decrease in penetration depth, which makes balancing resolution and depth challenging. Section title: Introduction Educational score: 4.006049633026123 Domain: biomedical Document type: Study Language: en The emergence of super-resolution ultrasound (SRUS) imaging technology has revolutionized breast cancer diagnostics. SRUS breaks the acoustic diffraction limit to improve spatial resolution, visualize microvascular, and detect low-speed blood flow ( 12 , 13 ). It can achieve up to ten-fold improvement in spatial resolution compared to conventional ultrasound imaging in theory ( 14 ). By locating individual microbubbles at the sub-wavelength level and tracking their displacement ( 11 , 15 ), SRUS creates super-resolution velocity maps (SRVM) to observe more microvascular details in breast masses. This technique has been successfully performed on humans ( 16 – 19 ). Moreover, based on microvessel density (MVD) and microvascular flow rate (MFR), it offers valuable information for medical diagnosis of breast cancer ( 20 – 22 ). Section title: Introduction Educational score: 4.004849910736084 Domain: biomedical Document type: Study Language: en Tissue elastic modulus, determined by tumor-associated matrix and collagen, plays a crucial role in the diagnosis and prognosis of tumor aggressiveness ( 23 ). Shear-wave elastography (SWE) is a technique that quantitatively measures tissue elastic modulus value and provides a potentially valuable tool to help differentiate benign from malignant breast masses ( 24 ).While SWE has demonstrated effectiveness in differentiating masses based on stiffness, it does not capture the microvascular characteristics critical for understanding tumor behavior ( 25 , 26 ). Section title: Introduction Educational score: 4.0938496589660645 Domain: biomedical Document type: Study Language: en By combining SRUS’s microvascular visualization with SWE’s quantitative stiffness measurements, our approach addresses the limitations of single ultrasound modalities that provide incomplete diagnostic information. Previous studies have explored the combination of B-mode ultrasound with SWE ( 5 ), SWE with CEUS ( 27 ), and B-mode ultrasound with CEUS ( 28 ) in breast cancer diagnosis. However, the combination of the tissue elasticity assessment of SWE and the microvascular imaging capabilities of SRUS are not well studied for breast cancer diagnosis, which distinguishes our study from prior work. Our study aims to develop a novel approach for the early diagnosis of breast cancer by integrating the microvascular and stiffness characteristics of breast masses. We also aim to investigate the correlation between Emax and MVD, and to assess the differential diagnosis efficacy of the combination of SRUS and SWE in benign and malignant breast masses, providing a more comprehensive diagnostic method and represents a meaningful contribution to the field. Section title: Patient population Educational score: 4.082425117492676 Domain: biomedical Document type: Study Language: en This prospective study was approved by the Institutional Review Board of China Resources & Wisco General Hospital. All the patients signed an informed consent form prior to the examination. 97 female patients (aged > 18 years) with breast masses were included in this study between October 2022 and May 2024. In cases where patients had more than one suspicious mass, the most suspicious-appearing mass was selected. Patients were excluded if they had contraindications to ultrasound contrast agents, a history of previous treatment, mental disorders, or any severe cardiovascular or cerebrovascular conditions. Additionally, those with hepatic or renal disease, or who were pregnant, were also excluded from the study. B-mode ultrasound, CEUS, and SWE examinations were performed for all patients. A SonoVue microbubble contrast agent (Bracco, Milan, Italy) was used. Furthermore, all patients underwent surgical excisions to obtain histopathologic results. Figure 1 illustrates the processes of patient registration and data processing. Section title: Clinical data acquisition Educational score: 4.009860038757324 Domain: biomedical Document type: Study Language: en The ultrasound examinations were conducted by a specific radiologist with over 15 years of experience. For real-time ultrasound image monitoring and data collection, a commercial ultrasound system (Resona R9T; Mindray Biomedical Electronics Co., Ltd., Shenzhen, China) and an L11-3U linear array probe with 3.0–10.0 MHz bandwidth were employed. Patients were provided with instructions to assume a supine position, raise their arms, and perform abduction movements during the scanning procedure to ensure comprehensive exposure of the breast and axilla. Furthermore, patients were instructed to maintain a state of quiet respiration during the examination. Section title: Clinical data acquisition Educational score: 4.1041669845581055 Domain: biomedical Document type: Study Language: en The probe was adjusted to show the maximum diameter of the breast mass and ensure that the surrounding tissues were clearly visible. After examining the sonographic characteristics of the breast mass with B-mode ultrasound, the probe was held perpendicular to the examination location, stabilized, and then switched to SWE mode. The whole mass and surrounding breast tissue were selected as two regions of interest (ROI) respectively, and the default values covered the range of 0–180 kPa. The Elasto function key was pressed on the control panel. The quality control badge in the upper right corner of the screen, which showed four stars or more, indicated satisfactory image quality. The image was frozen, and the mass contour was sketched manually along the sliding trackball by the radiologist. Each elastic modulus within the region was automatically calculated by the system, which stored the results as the maximum elasticity (Emax), minimum elasticity (Emin), mean elasticity (Emean), standard deviation of elasticity (Esd), and elasticity ratio (Eratio). Section title: Clinical data acquisition Educational score: 4.106417655944824 Domain: biomedical Document type: Study Language: en The SRUS image dataset was obtained from CEUS dynamic images after offline processing (see section 2.3 for details). An intravenous injection of a 0.5mL microbubble contrast agent was administered using a 19-gauge needle. The average frame rate was about 80 Hz. A mechanical index (MI) of 0.08 was used to avoid microbubble destruction during the CEUS examinations. The microbubble signal within the breast mass was observed and tracked using B-mode and CEUS dual-mode imaging. During imaging, patients were asked to hold their breath. Then CEUS images were gathered, and additional SRUS processing was performed on the CEUS images to facilitate the calculation of MVD and MFR as follows. Section title: Ultrasound image processing Educational score: 3.9790468215942383 Domain: biomedical Document type: Study Language: en SRUS image offline analysis was carried out using MATLAB software (MathWorks Inc., Natick, MA, USA). SRUS technology used ultrafast ultrasonic plane wave coherent composite imaging mode to collect the acoustic state of ultrasound microbubbles in microvessels continuously, established two-stage motion correction to correct tissue motion. Then, the post-processing of the SRUS algorithm was carried out. Section title: Ultrasound image processing Educational score: 4.133179187774658 Domain: biomedical Document type: Study Language: en SRUS used the acoustic response of the microbubble contrast agent to visualize the microvascular system. In this study, locating isolated microbubble signals first required singular value decomposition (SVD) to separate tissue and blood flow signals. The SVD method was first used to transmit and collect many pulse-echo signals and then carried out SVD to remove the tissue signals with large singular values to realize the extraction of microbubble signals. The spatial coordinates of the centroids of the microbubbles were extracted one by one by deconvolution of each source from the predicted Gaussian point spread function. The centroid location was to find the center of the microbubbles by calculating the intensity-weighted centroids of each extracted microbubble signal. With the superposition of multiple frames, SRUS rendering was realized. Section title: Ultrasound image processing Educational score: 4.059463024139404 Domain: biomedical Document type: Study Language: en MVD was defined as tracked microbubble area divided by the ROI area of mass. ROI was manually constructed on MATLAB based on breast mass contours on B-mode and SRUS images. To calculate the MFR, the tracking method calculated the best correlation bubble signal in the search window between adjacent images. Each microbubble detected in frame F and each microbubble in frame F+1 should be recorded in the search window. The study set the frame rate to 80 Hz and 700 microns as the maximum search window. For each signal in frame F, if the maximum normalized cross-correlation value in frame F+1 was higher than a determined threshold of 0.9, the pairing signal in frame F+1 was identified. Section title: Statistical analysis Educational score: 4.136686325073242 Domain: biomedical Document type: Study Language: en The Statistical Package for the Social Sciences version 26.0 (SPSS Inc., Chicago, IL, USA), the R Software (version 4.2.1; R Foundation for Statistical Computing, Vienna, Austria), and GraphPad Prism version 10.1.2 (GraphPad, Inc., San Diego, CA) were used to perform statistical analyses. T-tests, chi-square tests, and Mann-Whitney U tests were employed to compare statistical differences between the benign and malignant groups. Independent variables were screened, confounding factors were eliminated, and the remaining parameters were analyzed using multivariate binary logistic regression. Correlation analysis was conducted within this regression model, selecting variables significant in univariate analysis as independent variables to construct the binary logistic regression equation and determine the cutoff values. Receiver operating characteristic (ROC) curves and area under the curve (AUC) analyses were used to assess the diagnostic efficacy of SWE, SRUS, and their combined application for differentiating benign from malignant breast masses. Sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of the quantitative parameters were obtained via ROC analysis. The dataset was randomly divided into training and validation cohorts at a 7:3 ratio. The training cohort was used for binary logistic regression analysis to identify variables included in the nomogram, while the validation cohort was used for result validation. Nomograms and calibration plots were generated using the rms package. The rms package in R provides tools for building and validating regression models with an emphasis on survival analysis, logistic regression, and other predictive modeling techniques, as well as generating calibration plots and nomograms. The Hosmer-Lemeshow test was used to assess the model’s goodness-of-fit, and decision curve analysis (DCA), conducted with the rmda package, evaluated net benefits within the threshold probability range. The rmda package supports DCA, a method to evaluate the clinical utility of prediction models by calculating net benefits across a range of threshold probabilities. A scatter plot assessed the correlation between Emax and MVD. Statistical significance was defined as p < 0.05, with significance levels noted at 0.05 (*) and 0.01 (**) (two-tailed). Section title: Ultrasound images of breast masses Educational score: 3.952375650405884 Domain: biomedical Document type: Study Language: en The benign and malignant pathological spectra of the breast cases are shown in Figure 2 . Figures 3 , 4 showed B-mode, SWE, SRUS, and SRVM images of a patient with a benign and a malignant breast mass, respectively. The image below (e-h) corresponds to the local magnification in the box of the ultrasound image described above (a-d). SRUS demonstrated improved temporal and spatial resolutions while maintaining adequate penetration depth and visual field, enabling observation of microvascular at the micron scale. In the SRVM, red and blue indicated opposite directions and relatively high flow velocity, respectively, while yellow showed relatively low flow velocity, providing additional microvascular velocity information. Section title: Comparison of clinical information and quantitative parameters Educational score: 4.072470188140869 Domain: biomedical Document type: Study Language: en According to the data presented in Table 1 , the findings of this study demonstrated the presence of statistically significant variations in age, size, position, Emax, Emin, Emean, Esd, and MVD between benign and malignant masses ( p < 0.05), with malignant masses having greater values of Emax, Emean, Esd, and MVD, while benign masses had greater values of Emin. To further evaluate the diagnostic potential of these quantitative parameters, the study performed binary logistic regression analyses, and the results were presented in Table 2 . Only Emax and MVD showed statistical differences. The higher OR value of MVD compared with Emax indicated that MVD was more relevant in the differential diagnosis of breast masses. Section title: Relationship between Emax and MVD Educational score: 4.054154396057129 Domain: biomedical Document type: Study Language: en This research employed a scatter plot to assess the association between Emax and MVD. The scatter plot and fitting curve of correlation between Emax and MVD are shown in Figure 5 , and the result showed that Emax and MVD were significantly positively correlated, with a correlation coefficient of approximately 0.27 ( p < 0.01). Section title: Diagnostic performance of SWE and SRUS combination Educational score: 4.209977149963379 Domain: biomedical Document type: Study Language: en ROC analysis evaluated the diagnostic efficacy of SWE, SRUS, and the combination of SWE and SRUS in differentiating benign from malignant breast masses, including parameters such as sensitivity, specificity, accuracy, PPV, NPV, cutoff values, and AUC. Binary logistic regression analysis yielded regression equations for the SWE and SRUS groups as follows: for the SWE group, logit(p) = -1.857 + 0.036Emax - 0.225Emin; for the SRUS group, logit(p) = -1.112 + 0.454MVD, with cutoff values of 0.639 and 0.375, respectively. The regression equation and cutoff value for the combined SWE and SRUS group were logit(p) = -3.608 + 0.442MVD + 0.035Emax and 0.714, respectively. As presented in Table 3 , the SWE group demonstrated an AUC of 0.883 (95% CI: 0.815-0.952; p < 0.001), while the SRUS group had an AUC of 0.830 (95% CI: 0.745-0.914; p < 0.001). Notably, the combined SWE and SRUS approach yielded the highest AUC at 0.924 (95% CI: 0.875-0.973; p < 0.001), outperforming the use of SWE or SRUS alone. Consequently, the combined SWE and SRUS approach achieved an accuracy of 83.5%, sensitivity of 76.7%, and specificity of 94.6%. Figure 6 illustrates the ROC curves for SWE, SRUS, and their combined application, highlighting the superior diagnostic performance of the combined SWE and SRUS group with the largest AUC. Section title: Nomogram construction of SWE and SRUS combination Educational score: 4.1433186531066895 Domain: biomedical Document type: Study Language: en A multiparameter prediction model combining SWE and SRUS was developed and visualized as a nomogram to estimate breast cancer risk, as shown in Figure 7A . For example, consider a patient with the following parameter values: an Emax of 80.0 kPa (equivalent to 18 points on the nomogram), an Emin of 5.0 kPa (45 points), an Emean of 25.0 kPa (13 points), an Esd of 8.0 kPa (62 points), and an MVD of 1.5% (8 points). The cumulative score for this patient is 146 points. According to the nomogram, a total score of 146 points corresponds to an estimated breast cancer risk of 22%. Figure 7B The calibration curve for the nomogram demonstrated good agreement between the model’s predictions and actual observations. In both the training and validation cohorts, the Hosmer-Lemeshow test yielded p-values greater than 0.05 (p = 0.853 and p = 0.439), indicating a good fit of the nomogram to the data. The DCA of the nomogram is shown in Figure 7C . The results indicated that the SWE combined with SRUS model provided a higher net benefit in predicting breast cancer than either a treat-all-patients or treat-none approach across nearly all threshold probabilities in both the training and validation cohorts. Section title: Discussion Educational score: 4.183231353759766 Domain: biomedical Document type: Study Language: en The study involved 97 female patients with breast masses who underwent B-mode ultrasound, SWE, and CEUS examinations. The study included five distinct types of benign lesions and four distinct types of malignant lesions, categorized based on pathological subtypes. In our study, we evaluated the significance of the quantity parameters between SWE and SRUS, and explored the correlation between Emax and MVD. The results demonstrated that MVD exhibited the highest differential diagnostic value among the parameters evaluated, with a significant positive correlation to the Emax of the breast mass. Furthermore, we combined microvascular imaging quantification from SRUS with quantitative stiffness measurements from SWE to assess breast cancer and evaluated the diagnostic performance of this integrated approach. To evaluate the diagnostic accuracy, ROC curves, a nomogram, and DCA were utilized, highlighting the importance of these modalities in improving the diagnosis of breast masses and guiding clinical decision-making. The ROC analysis revealed that the diagnostic efficacy of the combined SWE and SRUS approach surpassed that of either modality alone. Additionally, DCA demonstrated that the SWE-SRUS combination model offered a higher net benefit in predicting breast cancer. Our integrated approach, combining SWE and SRUS, provides a more comprehensive diagnostic evaluation compared to conventional ultrasound methods. Section title: Discussion Educational score: 4.056377410888672 Domain: biomedical Document type: Study Language: en Previous studies have explored the combined diagnostic efficacy of various ultrasound modalities, such as B-mode ultrasound with CEUS ( 28 ), B-mode ultrasound with SWE ( 5 ), and CEUS with SWE ( 27 ). The integration of different ultrasound techniques has been shown to improve diagnostic accuracy. However, the potential benefits of combining tissue stiffness assessment through SWE with microvascular imaging from SRUS for breast cancer diagnosis remain unclear. Therefore, this study aims to evaluate the clinical applicability and potential of combining SWE and SRUS in the diagnosis of breast cancer. Section title: Discussion Educational score: 4.2179412841796875 Domain: biomedical Document type: Study Language: en SWE has been widely used to access breast masses based on tissue elastic modulus, with previous studies confirming its effectiveness in distinguishing between benign and malignant masses ( 29 – 31 ). In this study, the Emax, Emean, and Esd values of malignant breast masses were higher than those of benign masses, while Emin values were lower, consistent with earlier findings ( 32 – 34 ). This may be due to the presence of more elastic fibers and interstitial components in malignant masses, which are densely arranged. In contrast, benign masses often have a looser arrangement of fibrous stroma and glands, a higher mucopolysaccharide content, and a softer texture. For the microvascular quantification within breast masses, SRUS provides high resolution, and considerable depth, and surpasses the diffraction limit in structural imaging, making it applicable for differentiating benign from malignant breast masses. In this study, the quantitative analysis of SRUS parameters revealed that MVD was significantly higher in malignant breast masses compared to benign ones, while MFR did not differ significantly between the two groups. This may be due to malignant masses producing more growth factors, promoting angiogenesis, which increases MVD values. However, neovascularization in malignant tumors is often structurally and functionally abnormal, leading to vessel collapse and blood flow stagnation, which affects the flow rate ( 35 ). Section title: Discussion Educational score: 4.119852066040039 Domain: biomedical Document type: Study Language: en The value of MVD affects the biological behavior of breast cancer to some extent, and the biological behavior often determines the physical changes of the tumor, and the change of elastic modulus is also one of the physical changes of the tumor ( 36 , 37 ). Therefore, the elastic modulus may have some correlation with MVD, which is one of the primary purposes of this study. Because malignant masses usually have high heterogeneity, they exhibit significant regional differences in elastic modulus. And therefore Emax is a quantitative parameter which could better capture this heterogeneity, outperforming other elastic modulus values ( 38 , 39 ). This study primarily explores the correlation between Emax and MVD. This study found that Emax has a significant positive correlation with MVD, consistent with the conclusions of Jamin et al. and Jugé et al. ( 40 , 41 ). This may be because increased elastic modulus further activates signaling pathways related to tumor cell proliferation and invasion, thus, angiogenesis and vascular permeability are increased. MVD as a quantitative criterion of angiogenesis in breast tumors will increase accordingly ( 42 – 45 ). Section title: Discussion Educational score: 4.105201244354248 Domain: biomedical Document type: Study Language: en Our study showed that the AUC of SRUS combined with SWE was as high as 0.924, significantly higher than that of SRUS (0.830) or SWE (0.883). And it is worth noting that the combined group has higher specificity. These findings highlight that the combination of SRUS and SWE significantly improves diagnostic performance. ROC analysis showed that the AUC value of the SRUS group was slightly lower than that of the SWE group, which may be related to the selection of four statistically significant parameters Emax, Emin, Emean, and Esd in the SWE group. Only MVD was selected as a statistically significant parameter in the SRUS group. At the same time, the higher specificity of the combined group suggests that it can reduce false-positive cases and accurately exclude benign masses, which is of great value in reducing unnecessary biopsies. Lee et al. showed that combining quantitative parameters of SWE and superb microvascular imaging with B-mode significantly improved the diagnostic efficacy in differentiating benign from malignant breast masses, with an AUC of the combination of 0.849 ( 46 ). Chen et al. constructed a predictive model for breast cancer diagnosis using relevant parameters from SWE and CEUS. ROC curve analysis showed that the AUC reached 0.857, with significantly higher accuracy compared to conventional ultrasound ( 47 ). Our studies were consistent with the results of previous studies. This highlights the potential of this combined technique as an assessment of benign and malignant breast masses, thereby informing clinical decision-making and improving patient outcomes. Section title: Discussion Educational score: 3.836707592010498 Domain: biomedical Document type: Study Language: en We also developed a nomogram that combines SWE and SRUS to predict breast cancer risk, demonstrating strong performance on the calibration curve and in DCA. This nomogram shows potential clinical value by enhancing net benefit in clinical decision-making. Section title: Discussion Educational score: 4.053016662597656 Domain: biomedical Document type: Study Language: en Although this study included five types of common benign lesions and four types of common malignant lesions, with larger sample sizes and a broader pathological spectrum, the combined SWE and SRUS approach could offer valuable insights for identifying marginal or rare lesions. The approach incurs only the additional cost of an ultrasound contrast agent compared to conventional ultrasound, while offering significant potential to reduce unnecessary biopsies and associated healthcare expenses. Although a detailed cost analysis was not performed, the improved specificity of this method could help minimize misdiagnoses and follow-up interventions, ultimately reducing both patients’ psychological burden and overall healthcare costs. Section title: Discussion Educational score: 4.1200971603393555 Domain: biomedical Document type: Study Language: en However, this study has several limitations. The small sample size, limited range of breast disease types, use of only an internal validation cohort, and single-center design highlight the demand for larger sample sizes, a broader pathological spectrum, external validation, and multicenter prospective studies. In future research, we aim to optimize the study design, conduct multicenter prospective studies, and expand the cohort size to ensure more robust and generalizable findings, ultimately enhancing the clinical applicability of our results. Additionally, the two-dimensional (2D) nature of the ultrasound SRUS and SRVM images does not capture out-of-plane microvascular. Previous studies have reported three-dimensional (3D) ultrasound SRUS by acquiring 2D SRUS stacks using a mechanical scanning system to translate a one-dimensional array transducer ( 11 , 13 , 48 ). Advancements in 3D ultrasound SRUS might significantly enhance diagnostic accuracy by capturing the full volumetric context of breast masses, minimizing the risks of sampling bias inherent to 2D imaging. However, commercially available 3D super-resolution ultrasound equipment for clinical application has not yet been developed. To ensure consistency in image acquisition across different ultrasound modes, the maximum diameter section of each breast mass was selected for imaging. However, this approach may introduce potential sampling bias. For instance, local necrosis within the maximum diameter section could lead to decreased stiffness or reduced MVD. Notably, recent studies have demonstrated good repeatability of SRUS across different sections of breast masses ( 49 ), though the repeatability of SWE across various sections requires further investigation. Section title: Discussion Educational score: 4.045460224151611 Domain: biomedical Document type: Study Language: en In conclusion, the MVD of the breast mass exhibits a significant positive correlation with Emax. The combination of SRUS and SWE provides substantial advantages for breast cancer detection. This study presents an enhanced approach for the differential diagnosis of common benign and malignant breast masses, improving specificity and accuracy. Moving forward, we aim to further investigate the impact of SWE and SRUS on breast cancer histological types, biopsy rates, and other relevant factors. We believe that with continued research, the value of SRUS and SWE in the early diagnosis of breast cancer will become increasingly evident. | Other | biomedical | en | 0.999995 |
PMC11695222 | Section title: Introduction Educational score: 4.154295921325684 Domain: biomedical Document type: Study Language: en Trans fatty acids (TFAs), non-conjugated unsaturated fatty acids bearing one or more trans double bonds and presenting elaidic acid (9-trans-C18:1) as the primary isomer, are mainly obtained from hydrogenated vegetable oils and ruminant milk by humans ( 1 ). Epidemiological studies exhibit close correlations between TFAs intake and cardiovascular disease, atherosclerosis, diabetes, NAFLD or other metabolic diseases ( 2 , 3 ). Industrial TFAs can give rise to inflammation, endoplasmic reticulum stress, oxidative stress, and promote the preferential storage of fat in the liver, increasing the ratio of liver fat mass, steatosis, liver cholesterol levels, alanine aminotransferase activity, and fibrosis markers, indicating the enhancement of NAFLD ( 4 ). Section title: Introduction Educational score: 4.0001068115234375 Domain: biomedical Document type: Study Language: en NAFLD, a chronic liver metabolic disorder induced by abnormal lipid metabolism, includes fat infiltration in more than 5% of liver cells, non-alcoholic steatohepatitis (NASH), fibrosis, and liver cirrhosis ultimately leading to hepatocellular carcinoma, with a global prevalence of approximately 25% ( 5 , 6 ). NAFLD is associated with increased risks for liver related complications as well as cardiovascular diseases ( 7 ). Oleic acid can bind with triglycerides, cholesterol esters, phospholipids, and other long-chain fatty acids, leading to lipid metabolism disorders and increasing the potential risk for NAFLD ( 8 ). However, there is still limited evidence regarding the potential association between NAFLD and TFAs. Promoting lipid metabolism has become an effective means for treating obesity and NAFLD ( 9 ). Therefore, exploring the mechanisms of action for TFAs may provide a basis for treating diseases characterized by lipid metabolism disorders. Section title: Introduction Educational score: 4.2401123046875 Domain: biomedical Document type: Study Language: en Although some drugs have been employed for the treatment of NAFLD related metabolic disorders, they may bring some unpredictable side effects ( 10 ). As a member of the benzodioxolane family, sesamin is a fat-soluble furan lignan with molecular formula as C 20 H 18 O 6 and molar mass as 354.35 g/mol. Sesamin is mainly derived from sesame, and has also been found in more than 30 other medicinal plants ( 11 ). Sesamin exerts cholesterol lowering effects in serum and liver, as well as anti-inflammatory and antioxidant ones, and can alleviate lipid metabolism disorders ( 12 ). Sesamin can eliminate reactive oxygen species (ROS) and free radical induced damage to mitochondria, as well as promoting fatty acid oxidation to improve fat accumulation in the body ( 13 ). Shi et al. found that sesamin up-regulates the levels of proteins related to fatty acid oxidation, cholesterol efflux, and lipid metabolism via activating the AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor alpha (PPARα) signaling pathways, thereby reducing intracellular lipid accumulation during NAFLD ( 14 ). In addition, sesamin can improve hepatic steatosis and fibrosis in mice with NASH ( 15 ). However, further research is needed to determine whether sesamin can inhibit TFAs induced hepatic steatosis and the potential underlying mechanisms. Section title: Introduction Educational score: 4.17410945892334 Domain: biomedical Document type: Study Language: en Dysregulated lipid metabolism and oxidative stress are closely related to the occurrence and development of NAFLD, with autophagy playing a key role. The double membrane structure of autophagosomes isolates cytoplasmic substances and fuses with lysosomes, degrading the contents subsequently in lysosomes ( 16 ). Autophagy is necessary for regulating lipid metabolism since it could degrade lipid droplets ( 17 ). Therefore, autophagic disruption induced by steatosis may exacerbate liver lipid accumulation ( 18 ). Overexpression of liver specific autophagy related factor 7 (ATG7) induces autophagy and reduces steatosis and damage in NASH mice ( 19 ). However, the roles of sesamin in alleviating hepatic steatosis and oxidative stress, and activating autophagy has not been fully elucidated. Section title: Introduction Educational score: 4.325009346008301 Domain: biomedical Document type: Study Language: en The transcription factor EB (TFEB) has been identified as a major functional regulator promoting autophagy and lysosomal biogenesis at the transcriptional level ( 20 , 21 ). As the primary transcriptional regulator for autophagy lysosome pathway, TFEB positively regulates the expression of genes related to autophagy and lysosome biogenesis, thereby promoting autophagosome formation, autophagosome lysosome fusion, and degradation of autophagic substrates ( 22 ). TFEB also plays an important part in physiological processes such as lipid metabolism ( 23 ). Insufficient number of lysosomes and the declined hydrolytic enzyme activity are correlated with the occurrence of NAFLD ( 24 ). Upregulated TFEB can activate mitophagy and improve mitochondrial homeostasis ( 25 ). These findings emphasize the potential role of TFEB in treating NAFLD by promoting mitophagy dependent lipid degradation. However, it remains unclear whether TFEB plays a beneficial role in the regulation of hepatic autophagy flux and lipid degradation by sesamin. L02 cell line has become an important tool in scientific researches due to its high proliferation ability, preserved hepatocyte function, and specificity in response to exogenous factors. In the present work, L02 cells were employed as an in vitro model to investigate the alleviative effects of sesamin on steatosis induced by 9-trans-C18:1 and the underlying mechanisms ( 26 ). The results suggest that sesamin activates autophagy flux through the TFEB mediated autophagy-lysosome pathway, thereby reducing lipid accumulation. Section title: Materials Educational score: 1.3050739765167236 Domain: biomedical Document type: Other Language: en The Cell Counting Kit-8 (CCK-8) was provided by Meilun Biotechnology, with eltrombopag (EO) and MHY1485 purchased from MedChemExpress. The triglyceride (TG) kit (A110-1-1), trace malondialdehyde (MDA) kit (A003-1), and trace reduced glutathione (GSH) kit (A006-2-1) were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Improved Oil Red O staining kit , 4% paraformaldehyde , bicinchonininc acid (BCA) protein concentration determination kit , ROS detection kit , adenosine triphosphate (ATP) detection kit , mitochondrial membrane potential (MMP) detection kit , mitochondrial far-infrared fluorescence probe , Mito Tracker Green , Lyso Tracker Red , Hoechst 33342 , lipid droplet green fluorescence detection kit boron-dipyrromethene (BODIPY) 493/503 , cell autophagy staining (monodansylcadaverine (MDC) method) detection kit , along with adenovirus expression of monomeric Cherry (mCherry)-green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 beta (LC3B) fusion protein (Ad-mCherry GFP-LC3B) kit were purchased from Beyotime (Shanghai, China). The apoptosis detection kit was purchased from BD (Beijing, China). Section title: Cell culture and treatment Educational score: 4.0407209396362305 Domain: biomedical Document type: Study Language: en L02 cells (Shanghai Institute of Life, Chinese Academy of Sciences) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (supplemented with double antibiotics and nonessential amino acids) at 37°C under 5% CO 2 . Cells in exponential growth phase (3–10 passages) were seeded into various cell culture plates at moderate density for 24 h. The adherent cells were subjected to 9-trans-C18:1 and/or sesamin for 24 h. The study was divided into control, model, and intervention groups, respectively. Section title: Cell viability Educational score: 4.083643913269043 Domain: biomedical Document type: Study Language: en After L02 cells reach 80% coverage, they were digested and seeded into a 96 well plate. After adhesion, treatment with different fatty acids for 24 h was conducted. Then 10% CCK-8 medium were added into each well, followed by incubation at 37°C for 0.5–4 h. The absorbance at 450 nm was detected using a microplate reader (Multiskan GO, Thermo Fisher Scientific, Vantaa, Finland). Section title: Oil red O staining Educational score: 4.1403489112854 Domain: biomedical Document type: Study Language: en The cultured L02 cells were fixed with 4% paraformaldehyde at room temperature for 30 min and washed twice with phosphate buffered solution (PBS), respectively. Then, the cells were subjected to an appropriate amount of staining washing solution for 20 s and Oil Red O staining working solution for 20 min successively, followed by washing with washing solution and PBS for 30 and 20 s, respectively. The cell nucleus was stained with hematoxylin for 1 min. Microscopic observations were conducted using a microscope . After dissolving intracellular lipid droplets with 100% isopropanol, semiquantitative analyses were performed by measuring the absorbance at 510 nm using a microplate reader (Multiskan GO, Thermo Fisher Scientific, Vantaa, Finland). Section title: Ultrastructural observation Educational score: 4.0454535484313965 Domain: biomedical Document type: Study Language: en The treated L02 cells were digested and centrifuged at 2000 × g for 15 min. Subsequently, cell precipitates were collected and fixed with 3% glutaraldehyde fixative, dehydrated, infiltrated, embedded, ultra-thin sectioned, stained, successively. Finally, the ultrastructure was characterized using a transmission electron microscopy . Section title: Measurement of intracellular TG, MDA, and GSH Educational score: 4.052767276763916 Domain: biomedical Document type: Study Language: en The cultured and treated L02 cells were collected and washed twice with PBS. After being mixed with 300 μL PBS, the cells were sonicated in an ice water bath. The intracellular levels of TG (A110-1-1, Nanjing Jiancheng), MDA (A003-1, Nanjing Jiancheng), and GSH (A006-2-1, Nanjing Jiancheng) were detected according to the supplier’s instructions. A microplate reader (Multiskan GO, Thermo Fisher Scientific, Vantaa, Finland) was applied to measure absorbances at specific wavelengths. Section title: Measurement of intracellular ATP Educational score: 4.110641956329346 Domain: biomedical Document type: Study Language: en Intracellular ATP was quantified using a commercial reagent kit. After removing the culture medium, 200 μL of lysis buffer was added into each well of the 6-well plate to fully lyse the L02 cells. The supernatant was obtained by centrifugation at 4°C and 1.2 × 10 4 g for 5 min. After 100 μL ATP detection working solution was added into each detection well, 20 μL sample or standard was added and mixed quickly. The absorbance was measured using a multifunctional enzyme-linked immunosorbent assay reader (SpectraMax i3x, Molecular Devices, San Jose, United States). The concentrations of ATP in the samples were calculated based on the standard curve. Section title: Fluorescence staining Educational score: 4.128058433532715 Domain: biomedical Document type: Study Language: en After the medium removal, the L02 cells were washed once with PBS and incubated with 5 μM 2′,7′-dichlordihydrofluorescein diacetate (DCF-DA),1 × 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1), 200 mM Mito Tracker, and 1× MDC fluorescent probes prepared in serum-free 1640 medium at 37°C for 30 min. Then wash them once with PBS and medium, and replace with fresh medium. The intracellular ROS, MMP, mitochondria, and autophagy degrees were observed under a fluorescence microscope (DMi8-M, Leica, Germany). The semiquantitative analyses were performed using the Image J software. Section title: Apoptosis Educational score: 4.120630741119385 Domain: biomedical Document type: Study Language: en After removing the culture medium, L02 cells were washed once with pre-cooled PBS and then subjected to digestion with ethylene diamine tetraacetic acid (EDTA) free trypsin, washing, centrifuge, and resuspension in 1× Binding buffer. Each sample was added with 5 uL Annexin-V and PI in the dark and incubated at room temperature for 15 min. Then the apoptosis was analyze using a flow cytometer (FACS AriaIII, BD, United States) within 1 h. Section title: Mito-Lyso immunofluorescence co-localization Educational score: 4.066727161407471 Domain: biomedical Document type: Study Language: en Mito Tracker Green and Lyso Tracker Red were employed for the co-localization of mitochondria and lysosomes. After culture medium removal and washing once with PBS, L02 cells were added with Mito Tracker Green or Lyso Tracker Red storage solution diluted with culture medium at a ratio of 1:10000 and 1:13333, respectively, followed by incubation at 37°C for 30 min. After removing the staining solution and adding fresh culture medium, microscopic observations were carried out by a laser confocal microscope (TCS SP8, Leica, Wetzlar, Germany). Semiquantitative analyses were performed using the Image J software. Section title: Ad-cherry-GFP-LC3B adenovirus transfection Educational score: 4.032459259033203 Domain: biomedical Document type: Study Language: en After adhesion, L02 cells cultured in confocal dishes were transfected with Ad-mCherry-GFP-LC3B at a multiplicity of infection of 40 for 48 h and subsequently subjected to different treatment based on grouping. After removing old and adding fresh culture medium, they were observed using a laser confocal microscope (TCS SP8, Leica, Wetzlar, Germany). Semiquantitative analyses were performed based on yellow and red spots. Section title: Quantitative PCR analyses Educational score: 4.114755630493164 Domain: biomedical Document type: Study Language: en Total RNA was extracted from cells using TriZol reagent , followed by reverse transcription of mRNA into cDNA using a 1st strand cDNA synthesis kit . qPCR was performed on QantStudio 1 (Applied Biosystems, Walktham, MA, United States) using a 20 μL amplification system consisted of 10 μL premix (SsoFast EvaGreen Supermix, BioRad, CA, USA), 1 μL primer for each, 1 μL cDNA, and 7 μL deionized H 2 O. The qPCR reaction conditions were set as following: 95°C 30s for 1 cycle, along with 95°C 5s, 60°C 30 s, and 72°C 30 s for 40 cycles. Subsequently, melting curve analyses were performed to avoid amplification of non-specific products. β -actin was employed as an internal reference gene, and the mRNA relative level of the targets were calculated using the 2 -ΔΔCt algorithm. The primer sequences used in this work were presented in the Table 1 . Section title: Immunofluorescence staining Educational score: 4.183136940002441 Domain: biomedical Document type: Study Language: en After removing the culture medium, L02 cells cultured in confocal dishes were washed thrice with PBS and fixed with 4% paraformaldehyde at room temperature for 20 min successively, followed by washing thrice with PBS and 0.1% Triton X-100 addition. After being kept at room temperature for 10 min and washed thrice with PBS subsequently, they were added with protein free rapid blocking solution (PS108P, Epizyme, Shanghai, China) and blocked at room temperature for 30 min. After removing the blocking solution, an incubation overnight at 4°C with primary antibodies against TFEB and lysosome-associated membrane protein 1 (LAMP1) was performed. After washing thrice with PBS (5 min for each), the cells were incubated at room temperature for 1 h in the dark with Cy3 labeled sheep anti rabbit IgG (H + L) secondary antibody , followed by washing thrice with PBS (5 min for each). Hoechst 33342 was used for nuclear staining. Finally, microscopic observations were carried out using a laser confocal microscope (TCS SP8, Leica, Wetzlar, Germany). Section title: Western blotting Educational score: 4.157287120819092 Domain: biomedical Document type: Study Language: en The treated L02 cells were subjected to radio immunoprecipitation assay (RIPA) lysis buffer to extract total protein and a BCA assay kit for protein concentration measurement. Thirty μg protein was uploaded into each well and separate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane at a constant current of 300 mA, followed by block with 5% skim milk. Then the membrane was incubated with primary antibodies against microtubule-associated protein 1 light chain 3 (LC3) , PTEN-induced putative kinase 1(PINK1) , Parkin , Sequestosome 1(SQSTM1/p62) , TFEB , LAMP1 , glyceraldehyde-3-phosphate dehydrogenase (GAPDH) , and Histone H3 on a shaker overnight at 4°C. After being washed with tris-buffered saline with Tween 20 (TBST) (5 min for each), the membrane was incubated with HRP labeled sheep anti rabbit secondary antibody at room temperature for 1 h, followed by washing with TBST thrice (5 min for each). Finally, the protein bands were visualized using an ECL chemiluminescence assay kit in a chemiluminescence imaging instrument . The ImageJ software (NIH, Bethesda, MD, United States) was employed to analyze the densities of protein bands, with GAPDH acting as the reference gene. Section title: BODIPY 493/503 fluorescence staining Educational score: 4.136416435241699 Domain: biomedical Document type: Study Language: en Lipid droplets were stained using BODIPY fluorescent probe. After removing the culture medium, L02 cells cultured in a 24 well plate were washed twice with PBS and then fixed with 4% paraformaldehyde at room temperature for 15 min, followed by rinse twice with PBS. After adding 250 μL staining solution into each well, an incubation at room temperature in the dark for 20 min was performed. Hoechst 33342 was utilized for nuclear staining. After being washed twice with PBS, the cells were observed under a laser confocal microscope (TCS SP8, Leica, Wetzlar, Germany) and semi-quantitative analyses were performed using the Image J software. Section title: Statistical analyses Educational score: 3.10184645652771 Domain: biomedical Document type: Study Language: en All data were presented as means ± standard deviation (SD) and analyzed using the SPSS software (version 22.0, SPSS Inc., Chicago, Illinois, United States). One-way ANOVA (analyzes of variance) was employed for group comparison. LSD was applied for homogeneous variance and Dunnett T3 for the non-homogeneous. When p < 0.05, it was considered statistically significant. Section title: Sesamin alleviates lipid accumulation induced by 9-trans-C18:1 in L02 cells Educational score: 4.09482479095459 Domain: biomedical Document type: Study Language: en L02 cells were treated with 9-trans-C18:1 at various concentrations to detect its cytotoxicity. The results of CCK-8 showed that 9-trans-C18:1 at 500 μM exerted no significant effect on cell viability, and led to significantly increased intracellular TG ( p < 0.01), indicating the successful construction of the in vitro high-lipid model. In the absence or presence of 9-trans-C18:1 at 500 μM, sesamin at higher than 32 μM caused a significant decrease in cell viability . The results of Oil Red O staining (sesamin at 4, 8, 16, and 32 μM, respectively) showed a dose-dependent decrease in intracellular lipid accumulation . Therefore, 32 μM was selected as the working concentration of sesamin for subsequent experiments. These data indicate that sesamin reduced lipid accumulation in L02 cells treated with 9-trans-C18:1. Section title: Sesamin restores mitochondrial homeostasis by enhancing mitophagy Educational score: 4.320017337799072 Domain: biomedical Document type: Study Language: en The ultrastructure of L02 cells were analyzed through TEM. As shown in Figure 2 , the mitochondria in control group were oval and elongated, with clear cristae structures. Compared with control, intracellular lipid droplets in model group increased significantly, with significantly changed morphology and structure of mitochondria: higher proportion of slender mitochondria and blurred cristae. Partial mitochondrial cristae structures disappeared completely, showing shrinkage necrosis and vacuolization (Green arrow). Few mitochondria were engulfed by lysosomes and exhibited characteristic autophagic lysosomes (Red arrow), indicating low levels of cellular autophagy. Compared with the model group, intervention with sesamin resulted in a decreased volume ratio of intracellular lipid droplets by 51.3% , an increase in mitochondrial quantity, a restoration of oval and elongated structures, and a clear cristae structure, indicating an improvement on mitochondrial homeostasis . The increased lysosomes and autophagic lysosomes suggest an increase in cellular autophagy levels, conducive to timely removal of damaged mitochondria. The lipid toxicity caused by lipid accumulation may be the main reason for low-level cellular autophagy and premature degradation of damaged mitochondria. Therefore, it could be speculated that sesamin restores mitochondrial function by increasing mitophagy, thereby promoting lipid metabolism and reducing lipid accumulation. Section title: Sesamin improves oxidative stress and mitochondrial damage induced by 9-trans-C18:1 in L02 cells Educational score: 4.283571243286133 Domain: biomedical Document type: Study Language: en Subsequently, the impact of sesamin on cellular function was analyzed. ROS serves as an important indicator for cellular oxidative damage. Compared with control, the increased fluorescence intensity in the model group indicates an increase in intracellular oxidative stress level after 9-trans-C18:1 treatment, which could be alleviated by sesamin intervention, demonstrating its mitigating effect on intracellular oxidative stress . MDA can reflect the degree of lipid peroxidation in the body and indirectly reflect the degree of cell damage. GSH content is an important indicator for body’s antioxidant capacity. Sesamin significantly downregulated the intracellular MDA , while significantly upregulating the intracellular GSH , suggesting the role of sesamin in diminishing 9-trans-C18:1 induced oxidative stress level. The weakened mitochondrial MMP acts as the primary indicator for mitochondrial dysfunction. Compared with control, 9-trans-C18:1 treatment resulted in a significant decrease in intracellular MMP, which could be reversed by sesamin . The mitochondrial far-infrared fluorescence probe can detect cell apoptosis by detecting changes in MMP. The intensity of red fluorescence represents the function of mitochondria. As shown in Figure 3F . sesamin intervention enhanced the function of mitochondria, consistent with the MMP results. Decreased ATP levels usually indicates impaired or decreased mitochondrial function. The significantly reduced ATP levels in the model group were reversed by sesamin, indicating sesamin’s effects on restoring partial mitochondrial function . The above suggests that sesamin can improve the severe oxidative stress and disrupted mitochondrial homeostasis induced by 9-trans-C18:1 in L02 cells, thereby restoring partial mitochondrial function and diminishing lipid accumulation. Section title: Sesamin activates mitophagy through PINK1/Parkin pathway Educational score: 4.189266204833984 Domain: biomedical Document type: Study Language: en MDC is one of the most commonly used fluorescent probes for detecting cellular autophagy. Compared with control, autophagy in the model group was enhanced in some cells under 9-trans-C18:1 induction, with autophagy level relatively low, however. Compared with the model group, sesamin improved the cellular autophagy, thereby alleviating the 9-trans-C18:1 induced lipid accumulation to some extent and the degree of cell damage . Next, changes in mitophagy were examined through co-staining with MitoTracker and LysoTracker . Compared with control, the model group showed a significant decrease in mitochondrial fluorescence intensity, indicating that the accumulated lipid droplets significantly reduced mitochondrial homeostasis, in consistence with the TEM observation results . The fluorescence intensity of lysosomes and Mito-Lyso co-localization coefficient in the model group were significantly lower than those in the S32 group, indicating low mitophagy level in the model group. In the S32 group, significant enhanced Lyso fluorescence and Mito-Lyso co-localization coefficient were observed, suggesting that sesamin activated intracellular lysosomes and improved mitochondrial homeostasis by enhancing mitophagy levels, thereby reducing lipid droplet accumulation. As shown in Figures 4E , F , compared with the model group, the apoptosis rate of cells significantly decreased after intervention with sesamin, suggesting that sesamin may alleviate the degree of cell damage by enhancing autophagy levels to clear damaged mitochondria. Section title: Sesamin activates mitophagy through PINK1/Parkin pathway Educational score: 4.1595001220703125 Domain: biomedical Document type: Study Language: en PINK1/Parkin is a common mitophagy pathway which can be activated by mitochondrial dysfunction ( 27 ). Firstly, the mRNA levels of LC3 and p62 were detected by qPCR. Compared with control, the mRNA levels of LC3 and p62 were upregulated in the model group, which could be further upregulated by sesamin . Next, protein levels of LC3, p62, PINK1, and Parkin were analyzed using Western blot. As shown in Figures 5B , C , in comparison with control, the significantly increased protein levels of p62 suggest low-level autophagy under 9-trans-C18:1, consistent with the results of TEM , MDC , and Mito Lyso co localization . Compared with the model group, sesamin further significantly upregulated the protein levels of LC3, PINK1, and Parkin, while downregulated that of p62, indicating that the PINK1/Parkin pathway was activated, thereby promoting impaired mitophagy and improving mitochondrial homeostasis. Section title: Sesamin activates autophagy process by regulating TFEB nuclear translocation Educational score: 4.41737174987793 Domain: biomedical Document type: Study Language: en TFEB is activated during lysosomal impairment, which is part of the lysosomal injury response and necessary for subsequent lysosomal recovery, probably promoting lysosomal biosynthesis. The autophagy flux was analyzed through adenovirus transfection to effectively track the fusion process of autophagosomes and lysosomes. The results showed that the control cells exhibited diffuse yellow fluorescence of mCherry-GFP-LC3B with few autophagosomes and autolysosomes, indicating a non-autophagic state. Cells in the model group presented a large number of yellow spots and few red spots, suggesting that the cells attempted to initiate autophagy to alleviate the impairment caused by 9-trans-C18:1. Compared to the model group, a large number of red spots induced by sesamin suggest an increase in the fusion of autophagosomes and lysosomes and initiated process of autophagosome to autophagosomes to restore partial mitochondrial function, thereby alleviating the oxidative damage induced by 9-trans-C18:1, consistent with the results of TEM . Direct or indirect inhibitor of TFEB, EO or MHY1485, could reverse this trend. The mRNA levels of LAMP1 and TFEB were significantly upregulated before and after sesamin intervention, suggesting the up-regulating effects of both elaidic acid and sesamin on them . Compared with control, insignificant changes in the overall fluorescence intensity of TFEB but significantly elevated nuclear translocation rate in the model group were observed , indicating a role of 9-trans-C18:1 in dephosphorylating TFEB and inducing nuclear translocation partially. In comparison with the model group, the overall fluorescence intensity of TFEB was significantly enhanced and the translocation rate was significantly increased after intervention with sesamin, suggesting the activating effect of sesamin on TFEB translocation to initiate autophagy. EO or MHY1485 counteracted this effect. Compared with the model group, sesamin significantly upregulated the overall protein levels of TFEB and that in the nucleus , with that in the cytoplasm declined. EO or MHY1485 reversed this trend, consistent with the immunofluorescence results . Therefore, it could be speculated that sesamin promotes autophagy via regulating the translocation of TFEB. Section title: Sesamin activates autophagy process by regulating TFEB nuclear translocation Educational score: 4.169852256774902 Domain: biomedical Document type: Study Language: en The immunofluorescence staining of LAMP1 found that compared with control, the fluorescence intensity of LAMP1 in the model group was enhanced, indicating increased lysosomes to a certain extent after 9-trans-C18:1 treatment. The fluorescence intensity of LAMP1 was significantly enhanced by sesamin, which could be reversed by EO or MHY1485. This suggests that sesamin augments the count of lysosomes, thereby initiating autophagy and alleviating cellular impairment induced by 9-trans-C18:1. The Western blotting results exhibited upregulate the protein level of LAMP1 by sesamin, consistent with the immunofluorescence results. Section title: Sesamin activates autophagy process by regulating TFEB nuclear translocation Educational score: 4.180757999420166 Domain: biomedical Document type: Study Language: en BODIPY 493/503 probe can be used to characterize intracellular lipid droplets. The results showed that compared to control, the green fluorescence of the model group significantly increased. The fluorescence intensity was significantly diminished by sesamin. However, EO or MHY1485 could reverse the effect of sesamin. This suggests that sesamin may alleviate lipid accumulation induced by 9-trans-C18:1 through increasing mitophagy, thereby restoring partial mitochondrial function and mitigating the cellular impairment. As aforementioned, sesamin may activate the autophagy pathway regulated by TFEB via increasing the number of lysosomes, accelerating the process of autophagosome to autophagosomes to remove damaged mitochondria, thereby relieving the degree of cell damage induced by 9-trans-C18:1 and reducing lipid accumulation in L02 cells. Section title: Discussion Educational score: 4.1945576667785645 Domain: biomedical Document type: Study Language: en In the present work, the mitigative effects of sesamin on steatosis induced by 9-trans-C18:1 in L02 cells were investigated. The results showed that sesamin significantly reduced lipid accumulation in L02 cells by inhibiting oxidative stress, restoring mitochondrial morphology and function, and reducing cell apoptosis rate. Further analyses exhibited the involvement of autophagy in this process: sesamin may increase autophagy flux, promote nuclear translocation of TFEB and the expression of its target protein LAMP1, activate the autophagy-lysosome pathway to induce protective autophagy, thereby alleviating cell damage caused by 9-trans-C18:1 and reducing lipid accumulation in L02 cells. Section title: Discussion Educational score: 4.275498390197754 Domain: biomedical Document type: Study Language: en In recent years, more and more attention has been focused on dietary ingredients or natural products that can reduce liver steatosis and alleviate NAFLD ( 28 ). Sesamin can inhibit fatty acid synthesis, induce fatty acid beta oxidation, and promote the expression of genes related to cholesterol efflux and catabolism by activating the AMPK and PPARα signaling pathways, thereby alleviating lipid accumulation during NAFLD and preventing liver steatosis ( 11 , 14 , 29 , 30 ). Pham et al. reported that sesamin reduces palmitic acid-induced lipid toxicity in human hepatocellular carcinoma cell line (HepG2) and promotes liver lipid metabolism by activating the estrogen receptor alpha/calcium/calmodulin-dependent protein kinase β /AMPK signaling pathway ( 12 ). In addition, it was found that sesamin can improve liver steatosis and fibrosis in mice with NASH ( 31 , 32 ). Similarly, the present study found that sesamin can significantly reduce lipid accumulation induced by 9-trans-C18:1 in L02 cells, improve cell morphology and physiological function. It has been found that free fatty acid causes oxidative stress in hepatocytes, leading to lipid toxicity and hepatic steatosis. Sesamin can regulate lipid disorders by reducing lipid accumulation and inflammation in NAFLD ( 33 ). Here, it could also be found that sesamin significantly reduced intracellular ROS levels, apoptosis degree, and MDA, as well as enhancing GSH release levels. Sesamin indeed exerts antioxidant activity and protective effects on lipid accumulation in hepatocytes, while the underlying mechanisms needs further clarification. Section title: Discussion Educational score: 4.6000566482543945 Domain: biomedical Document type: Study Language: en Autophagy, a relatively conserved process in evolution, can transport cellular contents to lysosomes for degradation ( 34 ). Excessive TG and FFA in NAFLD patients can induce oxidative stress, leading to impaired autophagy ( 35 , 36 ). Impaired autophagy can lead to abnormal lipid accumulation in mouse liver and in vitro cultured hepatocytes ( 37 ), and blocking autophagy can also cause excessive lipid accumulation in the liver ( 38 ). Restoring autophagy through genetic or chemotherapy can alleviate hepatic steatosis in obese mice ( 39 , 40 ). Therefore, impaired autophagy can lead to the occurrence and progression of NAFLD ( 41 ). Enhancing autophagy is considered one of the most effective means to alleviate lipid accumulation and has significant implications for the treatment of NAFLD. In the present work, TEM results suggest that sesamin may alleviate lipid accumulation in L02 cells induced by 9-trans-C18:1 through activating mitophagy. The lipid toxicity caused by lipid accumulation may be the main reason for low-level autophagy and premature degradation of impaired mitochondria. Mitochondrial dysfunction can lead to lipid deposition in hepatocytes, causing hepatocytes damage and thus participating in the progression of NAFLD. Some scholars also believe that NAFLD is a mitochondrial disease ( 42 ). Mitophagy acts as one of the important means to remove dysfunctional mitochondria prior to activating apoptosis, by breaking down and reusing impaired mitochondria to maintain their normal structural stability ( 43 , 44 ). Therefore, sesamin might restore mitochondrial function in 9-trans-C18:1 treated L02 cells by increasing mitophagy, thereby promoting lipid metabolism and reducing lipid accumulation. The accumulated ROS enhances mitochondrial impairment, which is one of the factors triggering mitophagy ( 45 ). Park et al. found that the lipid toxicity induced by saturated fatty acids is mediated by excessive production of ROS, related to mitochondrial impairment ( 46 ). In addition, Liu et al. believe that declined MMP is a hallmark of mitochondrial dysfunction ( 47 ). Consistently, 9-trans-C18:1 treatment enhanced intracellular ROS production while reducing MMP, accompanied by inhibited ATP generation. Meanwhile, the far-infrared fluorescence representing the physiological activity of mitochondria also significantly weakened, indicating the occurrence of mitochondrial dysfunction. Interestingly, this process can be reversed by sesamin. After mitophagy was activated by various stimuli such as oxidative stress, levels of PINK1 and Parkin, p62 and autophagy related 16 like (Atg16L) protein, along with the autophagy marker LC3II/LC3I were correspondingly upregulated ( 48 ). Quercetin alleviates mitochondrial impairment and oxidative stress by enhancing Frataxin mediated PINK1/Parkin dependent mitophagy, thereby preventing liver lipid accumulation ( 49 ). Therefore, activating mitophagy may possess therapeutic potential in reducing hepatic steatosis. Section title: Discussion Educational score: 4.404166221618652 Domain: biomedical Document type: Study Language: en PINK1/Parkin dependent mitophagy is mainly involved in recognizing and clearing damaged mitochondria. Normally, PINK1 enters the mitochondrial inner membrane under the guidance of mitochondrial targeting sequences, and is subsequently cleaved by proteases on the inner membrane and released into the cytoplasm for hydrolysis. MMP decreases in the case of mitochondrial impairment, leading to the accumulation of PINK1 on the outer membrane of mitochondria. At the same time, Parkin undergoes phosphorylation and activation, selectively recruiting to damaged mitochondria. During this process, p62 is also recruited to mitochondria and then binds to LC3, mediating mitophagy ( 50 , 51 ). Knockout of mitophagy genes of Parkin and ATG7 exacerbated myocardial injury in high-fat induced mice ( 52 ). Section title: Discussion Educational score: 4.524133682250977 Domain: biomedical Document type: Study Language: en In the progression from NAFLD to NASH, the clearance of damaged mitochondria, inhibition of inflammatory mediators, and increased fatty acid oxidation are all associated with mitophagy ( 53 ). Overexpression of PINK1 can effectively restore mitophagy flux in aging macrophages ( 48 ). Decreased PINK1 in aging lungs leads to impaired mitophagy, promoting pulmonary fibrosis in young mice ( 54 ). Similarly, in this study, sesamin enhanced mitophagy and significantly reduced cell apoptosis rate. At the same time, sesamin significantly upregulated the ratio of LC3II/LC3I and protein levels of PINK1 and Parkin while downregulated p62 in L02 cells treated by 9-trans-C18:1, suggesting that sesamin indeed activates autophagy flux through the PINK1/Parkin pathway. Autophagy is a dynamic process, autophagosomes fuse with lysosomes and are subsequently degraded and cleared by lysosomes at the final stage of autophagic flux ( 55 ). Defects in lysosomal biogenesis and clearance inhibit autophagic flux, leading to lipid accumulation during the development of NAFLD ( 10 ). It has found that lysosomal biosynthesis in hepatocytes can be disrupted by high-fat diet (HFD), which is related to lipid metabolism ( 56 ). HFD induced hepatic steatosis can be alleviated by increasing autophagy flux ( 57 ). EO is a potent TFEB inhibitor and an effective and safe autophagy inhibitor ( 58 ). In addition, TFEB activity is also negatively regulated by the mechanistic target of rapamycin kinase complex 1 (mTORC1) ( 59 ), and mechanistic target of rapamycin kinase (mTOR) activation leads to TFEB phosphorylation in the cytoplasm and loss of activity. MHY1485, a mTOR agonist, can indirectly inhibit TFEB activation translocation to the nucleus ( 60 ). In this study, sesamin indeed enhanced autophagic flux employing EO and MHY1485. In addition, L02 cells initiated the process of autophagosome to autophagosome to alleviate oxidative damage induced by 9-trans-C18:1, thereby improving normal cellular morphology and function, restoring mitochondrial function partially, and alleviating cellular damage. EO and MHY1485 significantly reversed the increase in autophagy flux induced by sesamin. Therefore, it could be inferred that sesamin may activate autophagy inhibited by 9-trans-C18:1 in an autophagy-lysosome pathway dependent manner. Similarly, autophagy activation is beneficial and crucial for sesamin mediated alleviation of oxidative damage and lipid metabolism disorder induced by 9-trans-C18:1 in L02 cells. It has also been demonstrated that sesamin can activate the autophagy-lysosome pathway to alleviate lipid accumulation due to 9-trans-C18:1 treatment in L02 cells. However, further clarification is needed on whether sesamin activates the PINK1/Parkin autophagy pathway through TFEB. Section title: Discussion Educational score: 4.716958999633789 Domain: biomedical Document type: Study Language: en TFEB has been identified as the primary regulatory factor for autophagy and lysosomal biogenesis. Under normal conditions, TFEB exists in the cytoplasm in an inactive phosphorylated form, while fasting and stress conditions induce its dephosphorylation into the nucleus. Nuclear translocated TFEB can promote lysosomal biogenesis and substrate degradation ( 61 ), and induce expression of genes involved in lysosomal biogenesis ( 62 ). TFEB can promote the expression of genes related to lipid metabolism. In NAFLD patients, hepatic steatosis leads to TFEB phosphorylation mainly occurring in the cytoplasm. Therefore, TFEB activation may be an important marker for determining the degree of hepatic steatosis in NAFLD patients and associated with decreased autophagy activity ( 61 ). Overexpression of TFEB promotes autophagosome lysosome biogenesis, thereby inhibiting ethanol induced liver injury and steatosis in mice ( 63 ). Elevated levels of TFEB in the liver upregulates genes encoding lipid metabolism to oxidize FFA in mitochondria ( 64 ). The activation of TFEB-growth differentiation factor-15 (GDF15) in macrophages can regulate metabolic disorders such as obesity induced by HFD ( 65 ). The deficiency of TFEB in hepatocytes leads to a decrease in lipid metabolism, exacerbating metabolic diseases such as HFD induced hepatic steatosis and insulin resistance ( 66 ). There are also studies revealing that TFEB promotes nuclear translocation by activating the AMPK pathway and alleviates hepatic steatosis in mice fed with HFD ( 67 ). Here, it could be learned that sesamin can induce TFEB nuclear translocation and up-regulate its downstream target protein LAMP 1 to activate autophagy flux, and promote lysosomal biosynthesis. Therefore, sesamin probably promotes TFEB nuclear translocation and activates autophagosome-lysosome processes to reduce lipid accumulation in hepatocytes. Interestingly, TFEB activity may also be regulated by several key factors involved in autophagy, such as PINK1 and Parkin ( 68 ). Consistent with this, after treatment with sesamin, the mRNA levels of LC3 and p62, along with the ratio of protein LC3II/LC3I, significantly increased in L02 cells treated by 9-trans-C18:1. At the same time, levels of PINK1 and Parkin were enhanced, with p62 downregulated. Finally, it was found through BODIPY fluorescence staining that the lipid-lowering effect of sesamin can be reversed by EO or MHY1485. This suggests that sesamin may alleviate lipid accumulation via promoting intracellular mitophagy levels in a high-lipid in vitro model induced by 9-trans-C18:1, thereby restoring partial mitochondrial function and alleviating cell damage. Therefore, sesamin exerts its antioxidant stress and reduces lipid accumulation in cells by activating autophagy regulated by TFEB. However, the exact mechanism by which TFEB regulates autophagy in L02 cells still needs further clarification. Section title: Discussion Educational score: 4.138973236083984 Domain: biomedical Document type: Study Language: en This study revealed the mechanism by which sesamin exerts antioxidant stress and reduces lipid accumulation in L02 cells, and identified the important role of transcription factor TFEB in the autophagosome-lysosome pathway. However, our study also has some limitations. It has been reported that curcumin can induce Parkin dependent autophagy by activating AMPK and subsequent TFEB nuclear translocation, improve oxidative stress, enhance intestinal barrier function, and mitochondrial function ( 44 ). Therefore, the specific mechanism by which sesamin induces TFEB to regulate autophagy in L02 cells needs further clarification. In addition, it is necessary to explore other mechanisms by which sesamin induces autophagy in L02 cells. Finally, this study was only validated in an in vitro cell model and also needs to be validated in vivo . In our future work, it is necessary to explore more comprehensively underlying mechanisms by which sesamin regulates lipid metabolism. Section title: Conclusion Educational score: 4.073029518127441 Domain: biomedical Document type: Study Language: en Sesamin restores impaired mitochondrial morphology and function by activating the TFEB pathway, and significantly alleviates lipid accumulation induced by 9-trans-C18:1 in L02 cells, which may exert preventive or protective effects on hepatic steatosis. In the future, it is necessary to further explore the protective effects of sesamin on steatosis through multiple omics methods such as transcriptomics, proteomics, and metabolomics. | Study | biomedical | en | 0.999996 |
PMC11695226 | Section title: Introduction Educational score: 3.901538610458374 Domain: biomedical Document type: Study Language: en Pancreatic ductal adenocarcinoma (PDAC) is one the most lethal cancers, with an incidence rate of about 15 per 100 000 per year in Norway ( 1 ). About 85% of patients with PDAC are not eligible for upfront surgery with curative intent. Many of these receive neoadjuvant or palliative therapy, but unfortunately, the response rates are low ( 2 ). Biomarkers of chemotherapy response are crucial for selecting patients who are likely to benefit from treatment in a personalized way ( 3 ). Section title: Introduction Educational score: 4.367973804473877 Domain: biomedical Document type: Study Language: en In PDAC, carbohydrate antigen 19-9 (CA19-9) is the most commonly used biomarker for therapy response, but has limited predictive power ( 4 ). Considering new methods in oncology in general, tumor characteristics like expression of specific receptors or the presence of gene rearrangements have so far been the typical candidates as biomarkers ( 3 ). The gut microbiota composition has been increasingly associated with multiple health characteristics, including the response to some cancer therapies ( 5 , 6 ). Recently, Tintelnot et al. identified circulating levels of the gut microbial metabolite indole 3-acetate (3-IAA) as positively associated with response to chemotherapy in metastatic pancreatic ductal adenocarcinoma (PDAC) ( 7 ). In an elegant experimental and molecular characterization study, the mechanism of increased chemotherapeutic effect was found to depend on oxidation of 3-IAA by myeloperoxidase from neutrophils, which subsequently increased reactive oxygen species in tumor cells and hence tumor cell death. The study introduced a novel concept, whereby a metabolite produced by gut microbes is absorbed systemically and modifies chemotherapy response in the host ( 8 ). In the study, chemotherapy response in mice could even be improved by supplementing the diet with the 3-IAA precursor tryptophan, leading to increased 3-IAA levels, suggesting that a similar adjuvant approach may demonstrate clinical efficacy in humans. Section title: Introduction Educational score: 4.082162380218506 Domain: biomedical Document type: Study Language: en Chemotherapy in metastatic PDAC is given with palliative intent, but it is the primary treatment for patients with borderline resectable or locally advanced PDAC in order to improve resection rates with curative intent ( 2 ). We therefore measured 3-IAA in a cohort of patients with borderline resectable or locally advanced PDAC before starting chemotherapy ( 9 ), aiming to investigate if elevated serum 3-IAA associates with therapeutic response in a PDAC population without distant metastases. Section title: Study design and participants Educational score: 4.022612571716309 Domain: biomedical Document type: Study Language: en The participants included in this study were from the NORPACT-2 study ( 9 ), which was a prospective, population-based cohort of patients with borderline resectable or locally advanced PDAC enrolled at the Oslo University Hospital between 2018 and 2020. We included 124 patients who had donated blood before initiation of any chemotherapy . A subset of 35 (28%) patients who were available and willing to donate additional samples had a second blood draw 2–4 months after initiation of chemotherapy. The study protocol was approved by the Regional Ethical Committee of Medical and Health Research Ethics Nord 2017/1382. To assess population concentrations of 3-IAA, n=100 healthy control serum samples (median age 40, female 41%) studied in the context of other studies were also included ( 10 ), approved by the Regional Ethical Committee of Medical and Health Research Ethics South-Eastern Norway 2015/2140. Section title: Outcome, definitions and diagnostic criteria Educational score: 3.8908345699310303 Domain: biomedical Document type: Study Language: en Borderline resectable or locally advanced PDAC was diagnosed according to the National Comprehensive Cancer Network (NCCN) criteria, version 2, 2017 ( 2 ). Distant metastases were ruled out using computed tomography (CT) scans of the abdomen and chest. Fine-needle aspiration cytology or biopsy by endoscopic ultrasound was required to confirm PDAC. The chemotherapy regimen was decided by the treating medical oncologist at the local hospital. Section title: Outcome, definitions and diagnostic criteria Educational score: 3.634850025177002 Domain: biomedical Document type: Study Language: en The primary outcome was defined as overall survival, and time to the outcome was defined as the time from blood draw, which was done in conjunction with initiation of primary chemotherapy. A secondary outcome was included where participants were censored at their time of surgical resection (after start of primary chemotherapy) where applicable. Section title: Targeted liquid chromatography-tandem mass spectrometry Educational score: 4.043465614318848 Domain: biomedical Document type: Study Language: en Baseline and follow-up serum was sampled and stored according to a standardized procedure at Oslo University Hospital. Serum was left to coagulate for at least 30 minutes, followed by centrifugation at 2450 x g for 15 minutes and frozen and stored at -80°C. Targeted metabolite analysis of 3-IAA was performed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform, which is also used for measuring B-vitamins and human and bacterial tryptophan metabolites at BEVITAL ( www.bevital.no ) as described in Midttun et al. ( 11 ). Section title: Targeted liquid chromatography-tandem mass spectrometry Educational score: 4.125314235687256 Domain: biomedical Document type: Study Language: en Briefly, 3-IAA was added to the established assay using indole-2,4,5,6,7-d 5 -3-acetic acid (3-IAAd 5 ), obtained from C/D/N Isotopes Inc (Quebec, Canada), as internal standard. The retention times were 4.93 min (3-IAA) and 4.91 min (3-IAAd 5 ). The analytes were detected in positive-ion multiple reaction monitoring (MRM) mode, using the ion-pairs of 176.2/130.2 m/z and 181.1/134.1 m/z for 3-IAA and 3-IAAd 5 , respectively. Section title: Statistics Educational score: 4.050402641296387 Domain: biomedical Document type: Study Language: en Surviving proportions were visualized by plotting Kaplan-Meier survival curves of quartiles of 3-IAA. Cox proportional hazards models were used to estimate 3-IAA’s (log 2 -transformed) association with overall survival. We included ECOG performance status (modeled as a continuous variable), CA19-9 (log 2 -transformed), tumor classification (locally advanced or borderline resectable) and age in a multivariable Cox model. Section title: Statistics Educational score: 3.492795467376709 Domain: biomedical Document type: Study Language: en To test for differences in distributions of continuous variables we used the Wilcoxon rank-sum test for independent observations and the Wilcoxon signed-rank test for paired observations. Distributions are given as median (interquartile range, IQR). Section title: Statistics Educational score: 1.7747207880020142 Domain: biomedical Document type: Study Language: en For any variable used in the analyses, data were missing in less than 5% of the participants and missing data were therefore not imputed. All data processing, visualization and statistical analyses were performed in R. Section title: Results Educational score: 4.06442403793335 Domain: biomedical Document type: Study Language: en In total, 124 patients (median 68 years, 47% male) with borderline resectable (n=68) or locally advanced PDAC (n=56) were included ( Table 1 ). Seventy-six (61%) patients were treated with fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX), 26 (21%) with gemcitabine plus nab-paclitaxel, and 22 (18%) with gemcitabine monotherapy or other chemotherapy regimens. Section title: Results Educational score: 4.080282211303711 Domain: biomedical Document type: Study Language: en The median 3-IAA concentration before chemotherapy was 290 (IQR 203–417) ng/mL. This was within the range of concentrations observed in n=100 healthy individuals analyzed in the context of another study (median 377 (IQR 303–464) ng/mL). Serum 3-IAA was positively correlated with age but not with tumor diameter or serum CA19-9 . Pre-treatment 3-IAA was similar in the different chemotherapy groups . Section title: Results Educational score: 4.056593894958496 Domain: biomedical Document type: Study Language: en We found no statistically significant differences in pre-treatment 3-IAA concentrations according to response as defined by CT scan or CA19-9 decline . The 3-IAA concentration was numerically higher in patients who later had surgical resection and in those alive after one year, but the difference was not statistically significant . In participants with a second sample taken, 3-IAA concentrations were on average increased after chemotherapy (median 265 nmol/L before and 328 nmol/L after), but the increase was not statistically significant . Section title: Results Educational score: 4.112983703613281 Domain: biomedical Document type: Study Language: en The median survival time was 499 days (95% confidence interval (CI) 437–597). Twenty-six (21%) patients were event-free at their last follow-up date. We stratified participants into quartiles by pre-treatment 3-IAA concentrations but found no significant differences in survival between the groups , a finding supported by a Cox model where 3-IAA (log 2 ) had a HR=0.93, 95% CI [0.74–1.16], p=0.52. The model estimates were similar in the subgroups of borderline resectable or locally advanced cancer. Since 41 (33%) of the participants eventually had surgical resection of their primary tumor, which could influence survival, we evaluated the prognostic value of 3-IAA when censoring these cases at their time of surgery. Also here, we found no evidence of an association between 3-IAA and overall survival . Finally, using the full cohort, we adjusted 3-IAA for age, ECOG, CA19-9 and tumor classification, where 3-IAA (log 2 ) had an adjusted HR=0.87, 95% CI [0.68–1.12], p=0.28. Section title: Discussion Educational score: 4.009068965911865 Domain: biomedical Document type: Study Language: en In the present study, serum 3-IAA concentration was not associated with response to chemotherapy or overall survival in a population-based cohort of borderline resectable or locally advanced PDAC starting chemotherapy. The results suggest that high 3-IAA levels do not predict chemotherapy response in borderline resectable or locally advanced PDAC. Section title: Discussion Educational score: 3.983412027359009 Domain: biomedical Document type: Study Language: en The only data available for comparison are from the seminal paper by Tintelnot and co-workers ( 7 ) where an association between 3-IAA and chemotherapy response in PDAC was found. There are several differences between these studies that could potentially explain these conflicting observations. The association between chemotherapy response and 3-IAA in the Tintelnot et al. study was investigated in a total of 47 patients divided on two cohorts of metastatic PDAC ( 9 ). In contrast, while we studied more than the double number of patients (n=124), our population had no signs of distant metastasis. Patients with non-metastatic cancers often have a smaller total tumor load, better functional status ( 12 ), and differences in tumor biology compared with metastatic cancers ( 3 , 13 ), all of which may influence the effect of chemotherapy and its modifiers. Section title: Discussion Educational score: 4.086282253265381 Domain: biomedical Document type: Study Language: en Another major difference is the analytical methods. The median 3-IAA concentration in the present study was about 10-fold higher than what was found in Tintelnot et al. In a healthy control population analyzed in the context of other ongoing studies, the median 3-IAA was within the same order of magnitude. We therefore speculate that the discrepancy in 3-IAA concentrations arose mainly from assay differences, and it is difficult to exclude that this may in part contribute to the diverging results. Notably, the present data were generated with high quality LC-MS/MS-based methodology with a radioactively labelled internal standard. Nonetheless, if inter-individual variation in 3-IAA measurements was consistent with the two methods, we would expect to find an association with survival in our cohort if it truly existed. In our univariable model, 3-IAA had a wide confidence interval (0.74–1.16). While this makes it challenging to confidently assert the presence or absence of an effect, any potential clinical significance appears most likely to be small. Section title: Discussion Educational score: 3.975323438644409 Domain: biomedical Document type: Study Language: en A third point is that apparent predictive performances in discovery cohorts are often optimistic and models commonly perform worse in new populations for a variety of reasons ( 14 ). This is illustrated in the study from Tintelnot et al. where the effect size observed in the second human PDAC cohort was lower than in the first cohort (explained variance in the correlation between 3-IAA and survival time was R 2 = 0.51 in a cohort from Hamburg and R 2 = 0.24 in a cohort from Munich). Section title: Discussion Educational score: 3.8000471591949463 Domain: biomedical Document type: Study Language: en One of the imitations of our study is that 54% of the patients received a biliary stent due to jaundice, and it remains unclear whether hyperbilirubinemia influences the gut metabolism of tryptophan, the precursor of 3-IAA ( 15 , 16 ). Furthermore, only 28% of the cohort had a second blood sample withdrawn for serum 3-IAA measurement and only 61% were treated with FOLFIRINOX. Section title: Discussion Educational score: 3.953223943710327 Domain: biomedical Document type: Study Language: en In conclusion, our results do not align with the optimism generated by Tintelnot et al.’s study. Additional validation of 3-IAA as a biomarker of chemotherapy response in PDAC is necessary before initiating human clinical trials aiming to modify 3-IAA levels in this setting. Furthermore, subsequent research in this field should probably focus on metastatic PDAC. | Study | biomedical | en | 0.999996 |
PMC11695230 | Section title: Introduction Educational score: 4.134252548217773 Domain: biomedical Document type: Study Language: en Clomazone (Clo), an isoxazolane herbicide, was primarily employed for the control of annual grass and broadleaf weeds in crops such as peanuts, soybeans, and oilseed rape through pre- and post-emergence applications . So far, there are over 200 clomazone-based products in China, mostly used for soil treatment . The applied clomazone do not completely degrade over time, with a major portion remaining in the soil as residues . The half-life of clomazone in soil ranges from 15 to 157 days, and its biological activity lasts for over 180 days, making it a persistent environmental contaminant . For example, the Mollisols region of China’s cropland soils contained 56 detected herbicides, with residues ranging from 0.31 to 1558.13 μg/kg. Clomazone exhibited the highest concentrations . The high solubility and low log Kow (2.55) of clomazone make it easily absorbed by subsequent sensitive crops, causing phytotoxic symptoms like foliar bleaching, reduced plant height, and decreased yields . Therefore, it is essential to assess the mitigation of residual damage caused by Clo in maize for the safe production of maize. Section title: Introduction Educational score: 3.9962384700775146 Domain: biomedical Document type: Review Language: en Safeners are chemical agents added to herbicide formulations to protect crops from potential harm without reducing the effectiveness of the herbicides against targeted weeds, allowing farmers to apply them more safely and effectively while minimizing crop injury and achieving desired crop protection . The number of safener utilized in commercial herbicide formulations currently stands at approximately 20. Safeners are commonly applied as seed treatments (e.g., Naphthalic anhydride, Cyometrinil, Oxabetrinil, Fluxofenim, Flurazole in maize or sorghum), water surface applications (Dymron, Cumyluron, Dimepiperate in rice), or spray formulations (Benoxacor, Furilazole, AD-67, Fenclorim used for pre-emergence in maize; Cloquintocet-mexyl, Fenchlorazole-ethyl, Mefenpyr-diethyl, Isoxadifen-ethyl used for post-emergence in cereals or maize) . Cyprosulfamide (CSA) was launched by Bayer CropScience AG in 2009 and is the only safener that can be used for pre- and post-emergence herbicides in maize . It can effectively mitigate damage to maize caused by isoxaflutole , thiencarbazone-methyl , fenpyrazone , and nicosulfuron . The majority of studies have focused on the efficacy of CSA in alleviating herbicide phytotoxicity, with limited research conducted on residual herbicide damage. Section title: Introduction Educational score: 4.446605682373047 Domain: biomedical Document type: Study Language: en The previous research has shown that safeners effectively reduce the negative impacts of herbicide residues. N-tosyloxazolidine-3-carboxamide derivatives, compound 9 (N-phenoxyacety-2-methyl-2,4-diethyl-1,3-oxazolidine) showed significant protection against tribenuron-methyl and nicosulfuron via enhancing the glutathione (GSH) content and glutathione S-transferase (GST) activity . The tolerance of crops is enhanced by safeners through two primary mechanisms. First, the amount of herbicide reaching the active site may be reduced or its interaction at the target site may be disrupted . Second, the activation of detoxifying enzymes by safeners plays a crucial role in the metabolism of herbicides . The focus of most studies on detoxifying enzymes was glutathione S-transferases (GSTs), which catalyze the conjugation of herbicides with endogenous glutathione in wheat, maize , and rice . Other classes of detoxifying enzymes, such as cytochrome P450s (CYP450s), UDP-dependent glycosyltransferases (UGTs) , ATP-binding cassette transporters (ABC transporters) and antioxidant system (SOD, POD, CAT) , are also involved in the process of detoxification. The previous investigation have confirmed that CSA effectively alleviate the damage caused by nicosulfuron on maize plants under adverse environmental conditions, such as high temperatures and dry weather, while simultaneously enhancing herbicidal efficacy against weeds . The mitigation of nicosulfuron damage was associated with the enhancement of nicosulfuron metabolism through the up-regulation of genes involved in the detoxification pathway . We propose that these metabolic enzyme genes play a crucial role in the mitigation of Clo toxicity by CSA. Section title: Introduction Educational score: 4.164734363555908 Domain: biomedical Document type: Study Language: en The present study objectives to investigate mitigating effect and the mechanism of the mitigation of Clo residual damage by CSA. The compounds known as CSA and Clo are both biologically active xenobiotic substances. As herbicides can induce peroxidation in crops, the impact of safeners on ROS in maize remains uncertain. The effects of CSA on maize physiology and the expression of metabolic enzyme genes in maize under the influence of CSA and Clo are systematically studied. Here, we aim to (1) investigate the potential of CSA in enhancing chlorophyll and GST activity, (2) explore the antioxidant enzyme activities in maize seedlings under Clo and CSA treatment and (3) identify genes closely related to the mode of action of the CSA and Clo metabolic pathways. This will address the issue of residual damage caused by Clo. It also provides a theoretical framework for investigating the mechanism of CSA. The combined use of these methods not only improves crop protection and yields, but also helps to make agricultural practices more sustainable by reducing environmental risks. Section title: Plant material and growth conditions Educational score: 1.6282418966293335 Domain: other Document type: Other Language: en The soil for the test was loamy soil from the experimental base of Henan Academy of Agricultural Sciences, and the physical and chemical properties of the soil are shown in Table 1 . The maize seed used for the test crop was Zhengdan 958, purchased from Henan Qiule Seeds Technology,Co.,Ltd, Henan Province, China. The herbicide safener CSA with 94% purity was purchased from Hebei Lansheng Biotech Co., LTd, Hebei Province, China. Clomazone (360 g L -1 ) was produced by Yunfa Chemical Factory (Shanghai, China) Co. Section title: Plant material and growth conditions Educational score: 4.102042198181152 Domain: biomedical Document type: Study Language: en Maize seeds were soaked in distilled water for 2 h, followed by germination at a temperature of 28°C in the dark for 48h. when the radicle length of maize reached approximately 0.5cm, the maize were subjected to immersion in water (CK) or 120 mg/L CSA solutions for 2 h. Afterward, the seeds were washed three times with distilled water. Subsequently, they were planted in 7 × 8 cm (diameter × height) square nutrient pots, with five seeds per pot. The pots contained soil supplemented with Clo at a concentration of 4 mg/kg (the concentration selection process is given in the Supplementary Data Sheet ). Seeds were sown to a depth of 3 cm. All pots were incubated in an artificial greenhouse (20 to 25°C, L//D = 12 h//12 h, and 80% relative humidity). Five biological replicates were performed for each treatment. Plant height and fresh weight were measured 7 days after seedling emergence. The shoots of maize under different treatment were collected 7 days after emergence for chlorophyll, malondialdehyde (MDA), GST, reduced glutathione (GSH), oxidized glutathione (GSSG), antioxidant activity analyze and qRT-PCR determination. Section title: Physiological indicators and chlorophyll content determination Educational score: 4.119335174560547 Domain: biomedical Document type: Study Language: en Total chlorophyll (chlorophyll a, chlorophyll b, carotenoid and total chlorophyll) was extracted by 80% acetone and the content assays was detected using spectrophotometric methods . GST, GSH and GSSG were extracted and quantified using determined by a kit (Grace, Suzhou, China) in accordance with the manufacturer’s instructions. Protein content was determined by Bradford’s koji method . The crude enzyme solution for determined SOD and CAT activity was extracted in accordance with the methodology proposed by Nayeri et al. . SOD enzyme activity was determined by the nitroblue tetrazole (NBT) method ; CAT activity was determined by UV absorption . The MDA content was determined by thiobarbituric acid colorimetric method . Section title: RNA extraction and real-time fluorescence quantitative PCR assay Educational score: 4.113715171813965 Domain: biomedical Document type: Study Language: en The transcriptom data published by Sun et al. was utilized to identify a total of twenty-eight ( Supplementary Table S1 ) maize detoxification genes that were up-regulated in response to CSA treatment. Based on the respective gene sequences, candidate gene-specific primers were designed using Primer Premier 5.0 and synthesized by Sangon Biotech CO., Ltd. (primers are shown in Supplementary Table S2 ). Leaf tissue samples stored at -80°C were ground in liquid nitrogen and total RNA was extracted using Vazyme reagent (catalog No. RC411-01; Vazyme Biotech Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. Quantitative real-time quantitative polymerase Chain reaction amplification templates were 1 μL of cDNA, 0.4 μL (10 μM) of each primer, 5 μ of TB Green premix Ex TaqII (Takara, Beijing, China), and 3.2 μL of distilled water. According to our previous methods, EF1α was used as a reference gene . CK was used as the control tissue, and the relative quantification of the gene in each tissue was determined by the comparative 2 -ΔΔCT method. Section title: Statistical analysis of data Educational score: 2.9173405170440674 Domain: biomedical Document type: Study Language: en The data are presented as mean ± standard deviations. The significance of the treatment differences was assessed using one-way analysis of variance (ANOVA) based on the least significant difference (LSD) at a significance level of P < 0.05 level, employing the SPSS software 26.0. The graphs were plotted using GraphPad Prism 9. Section title: The protective effect of CSA on maize against Clo residue injury Educational score: 4.147292137145996 Domain: biomedical Document type: Study Language: en The economic crop maize, often rotated with soybeans, frequently suffers from clomazone residue-induced injury . The toxicity of Clo in maize was investigated under both CSA-treated and non-treated conditions. After 7d of treatment, the visible symptoms of Clo include leaf chlorosis, wilting, and inhibited plant growth . Maize plants showed a 19% inhibition in plant height and a 29.9% reduction in fresh weight . However, the CSA-pretreatment significantly protected maize from Clo exposure and suppressed its growth inhibition. The bleached maize leaves started to regain their green color . The inhibition of plant height and fresh weight of maize was reduced by 9.4% and 7.2% . The CSA-pretreatment alone did not significantly affect maize fresh weight. The results confirmed that enhancing Clo detoxification in maize plants correlated with a protective effect, which depended on soaking CSA 2 hours before Clo treatment. Section title: Enhanced chlorophyll content in maize leaves after pre-treatment with CSA Educational score: 4.225438117980957 Domain: biomedical Document type: Study Language: en Clo inhibits 1-deoxy-D-xylulose-5-phosphate (DXP) synthase in the first committed step of the non-mevalonate isoprenoid pathway in plastids, causing impaired chloroplast development and loss of pigments including carotenoids in susceptible plants . To assess the impact of CSA on maize leaf chlorophyll content, the chlorophyll and carotenoid content in maize leaves was determined following CSA treatment. The content of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid of maizes in CSA-treated maize leaves show no significant effect compared to those in the control treatment . The content of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid in maize plants decreased by 92.9%, 91.2%, 92.5% and 87.0% respectively under Clo treatment compared to the control . This may be related to the mechanism of Clo. The treatment with soaking CSA significantly mitigated the impact of Clo on chlorophyll a, chlorophyll b, total chlorophyll and carotenoid, resulting in an increase in content by 8.09, 6.05, 7.62 and 4.69 times, respectively. These results suggest that the pre-treatment with CSA may be increased the rate of Clo metabolism in maize plants. Section title: Pre-treatment with CSA decreased MDA content and enhanced SOD and CAT activity Educational score: 4.06479024887085 Domain: biomedical Document type: Study Language: en The activity of antioxidant enzymes has been shown to be associated with herbicide tolerance in various plants . To assess cellular damage induced by Clo and CSA, MDA content in maize plants was quantified. As shown in Figure 3C , The MDA content of maize increased significantly by 5.3 times with Clo treatment alone compared to the control. However, CSA+Clo treatment reduced the MDA content by 13.44% compared to Clo treatment alone. The result suggests that CSA may potentially mitigate the accumulation of MDA induced by Clo. Section title: Pre-treatment with CSA decreased MDA content and enhanced SOD and CAT activity Educational score: 4.101852893829346 Domain: biomedical Document type: Study Language: en The Clo and CSA treatments resulted in alterations to in superoxide dismutase (SOD) and catalase (CAT) activities in maize . The activity of SOD and CAT decreased by 46.80% and 31.04%, respectively, under Clo treatment compared to the control. However, when CSA+Clo treatment was applied, SOD and CAT activities increased by 9.7% and 11.4%, respectively, compared to Clo treatment alone . The capacity of the addition of CSA to reduce the level of MDA may be attributed to its potential to enhance the activities of the enzymes SOD and CAT. Section title: Pre-treatment with CSA increased GSSG content and enhanced GST activity Educational score: 4.130545616149902 Domain: biomedical Document type: Study Language: en The enzyme GST plays a crucial role in helping crops detoxify herbicides by facilitating the binding of GSH with various types of herbicides . The Clo residue treatment significantly inhibited GST activity and decreased GSH and GSSG content in maize seedlings. Specifically, there was a reduction of 50.80% in GST activity, 27.27% in GSH content, and 77.13% in GSSG content compared to the control group. However, this inhibition was alleviated after CSA+Clo treatement, with GST activity and GSSG content increasing by 26.7% and 31.3% compared to Clo treatment alone . GSH content was no significant effect between the Clo and Clo+CSA treatment. The results indicate that CSA enhances detoxification and metabolism of Clo in maize seedlings, improving their tolerance to Clo. Section title: Metabolizing enzyme genes expression Educational score: 4.141085624694824 Domain: biomedical Document type: Study Language: en The expression of specific genes encoding detoxification enzymes in crops can be induced by safeners . We previously identified several detoxification enzymes genes in maize induced by CSA through transcriptome . These CSA-upregulated genes are predicted to play a crucial role in mitigating the residual injury of Clo. Suitable primers were designed for 24 out of the 28 CSA-induced metabolizing enzyme genes, but CYP72A14 and UGT88A1 showed down-regulation in the CSA treatment, which contradicted the transcriptome data (refer to Supplementary File for details), leading to their exclusion. Finally, 22 candidate genes were screened, namely, CYP72A5 , CYP81A4 , CYP81Q32 , CYP81A9 , CYP81A36 , UGT83A1 , UGT88A1 , UGT76C2 , ABC , GSTIV , GST30 , GST19 , GSTZ5 , GST31 , GST4 , GST25 , GST39 , GST21 , GST37 , In2-1 , GSTVI , GST7 and GSTU6 . The expression levels of all 22 candidate genes were significantly up-regulated after CSA treatment alone, ranging from 1.05 to 42.52 in relative expression . The result aligns with the findings reported by Sun et al. . However, the responses of these 22 candidate genes varied under Clo and CSA+Clo treatments. Section title: Metabolizing enzyme genes expression Educational score: 4.099517822265625 Domain: biomedical Document type: Study Language: en As shown in Figures 5B, C, E , the expression levels of three P450 genes ( CYP81A9, CYP81Q32 , and CYP72A5 ) remained unchanged under Clo treatment. However, when treated with CSA+Clo, the expression of these three genes significantly increased by 10.56-, 4.98-, 6.74-fold compared to that in the Clo treatment. On the other hand, the relative expression level of CYP81A36 and CYP81A4 were 0.22 and 0.17, respectively, under the Clo treatment . Their expression levels increased remarkably by 25.67- and 10.27- fold respectively under Clo+CSA treatment in comparison to Clo treatment. Section title: Metabolizing enzyme genes expression Educational score: 4.1702680587768555 Domain: biomedical Document type: Study Language: en As shown in Figure 6 , GST30 , GST31 , GSTU6 , GSTIV , GSTVI , GST21 , GST7 , GST37 , ABC and UGT83A1 showed no significant change in response to Clo treatment . However, the expression of these ten genes significantly high increased by 16.70-, 46.92-, 0.81-, 7.53-, 5.10-, 238.82-, 143.50-, 4.58-, 5.67-, 10.47-fold after CSA+Clo treatment compared to that in the Clo treatment. The expression of the GST4 gene was significantly increased by 1.68 times under Clo treatment, while it was increased by 3.97 times under CSA+Clo treatment . Conversely, the expression of the GST19 , GST25 , GST39 , GSTZ5 , In2-1 and UGT76C2 genes showed a significant decrease after Clo treatment, with reductions of 75.32%, 97.09%, 39.17%, 71.26%, 78.56% and 68.90%, respectively . However, when treated with CSA+Clo, there was a notable increase in gene expression compared to Clo treatment: a fold increase of 1.71 for GST19 , 31.51 for GST25 , 0.87 for GST39 , 1.95 for GSTZ5 , 39.30 for In2-1 and 4.20-fold increase for UGT76C2 . The gene expression analysis revealed an up-regulation of genes related to Clo detoxification pathways in maize pre-treatment with CSA. Section title: Discussion Educational score: 4.248718738555908 Domain: biomedical Document type: Study Language: en The findings of our study showed that CSA effectively mitigated the adverse effects of Clo on maize seedlings, as evidenced by improvements in plant height and fresh weight . Additionally, it alleviated the decrease in chlorophyll and carotenoid content . This finding is consistent with the results of previous studies which demonstrated that CSA was able to alleviate the residual damage caused by nicosulfuron . The carotenoid and chlorophyll contents in maize plants are increased when pre-treatment with CSA, even when the MEP (methylerythritol 4-phosphate) pathway is inhibited by Clo. Reactive oxygen species (ROS) are associated with herbicide toxicity, leading to membrane damage through lipid peroxidation . Previous studies indicated that when herbicides are absorbed by crops, the activity of SOD in leaves increases, maintaining the levels of reactive oxygen free radicals at a relatively low level. When it is not sufficient to clear the superoxide anions caused by herbicide toxicity, the SOD activity decreases and the accumulation of oxidative substances increases, which may cause damage to the cell membrane system . This study showed that Clo significantly elevated MDA levels while reducing SOD and CAT activities, as it can cause serious harm to maize seedlings. On the contrary, pre-treatment with CSA enhanced Clo-induced oxidative stress and reduced MDA accumulation in maize seedlings. However, CSA alone did not elevate SOD and CAT levels. This suggested that the defensive antioxidative properties are also one manner in which CSA protects maize from injury caused by Clo residue. In a previous study examining the effects of chloroacetamide herbicides and safeners (mefenpyr and dichlormid) on human blood cells, it was found that while the safeners alone did not cause any changes in oxidative stress, they did reduce the lipid peroxidation induced by the herbicides . Section title: Discussion Educational score: 4.225810527801514 Domain: biomedical Document type: Study Language: en The induction of glutathione S-transferases (GSTs) by safener plays a crucial role in the phase II detoxification system . Chronopoulou et al. observed a strong correlation between the efficacy of safeners and their capacity to induce GST activity. The present study demonstrated that Clo alone or co-application with CSA was reduces GST activity GSH and GSSG content compared to CK. Pre-treatment with CSA for two hours, the GST activity and GSSG content significantly increased, this indicate that the GST enzyme facilitates the binding of GSH with the herbicide Clo, resulting in the formation of the CLO-GSH conjugate and an increase in the oxidation state of GSSG. Similarly, the safener isoxadifen-ethyl has been shown to improve maize tolerance to the toxic effects of nicosulfuron by increasing the activity of this enzyme in maize . Similar findings have been reported in other species. The study by Hu et al. showed that fenclorim enhances rice crop protection against pretilachlor herbicide by increasing GST enzyme activity. The results of this study, along with previous findings, suggest that CSA can enhance GST activity and mitigate herbicide-induced damage. Section title: Discussion Educational score: 4.264232635498047 Domain: biomedical Document type: Study Language: en The molecular mechanism of safener may involve complex interactions among multiple pathways that protect plants from herbicides and other compounds . Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was employed to examine the gene expression of detoxification enzymes, including P450, GST and UGT, in response to CSA alone or in combination with Clo treatment . Cytochrome P450 monooxygenase plays a role in the metabolism of herbicides. In this study, the expression of five genes, namely CYP81A9 , CYP81A4 , CYP81A36 , CYP72A5 , and CYP81Q32 , was no effect or significantly down-regulated by Clo residue treatment, but their expression were significantly enhanced by the pre-treatment with CSA . These five P450 genes may be related to metabolism of Clo. Among them, CYP81A9 plays a crucial role in the hydroxylation process of CSA and detoxification of nicosulfuron in maize . CYP81A4 has the potential to limit the activity of bentazon thiadiazine . This suggested CYP81A9 and CYP81A4 may be involved in the metabolism of Clo, but CSA itself can also be metabolized by P450. Section title: Discussion Educational score: 4.321725845336914 Domain: biomedical Document type: Study Language: en The second phase of herbicide metabolism is primarily catalyzed by two crucial enzyme families, GSTs and UGTS . Nine GST genes ( GST30 , GST31 , GSTIV , GSTVI , GST21 , GST7 , GST37, GST25 , IN2-1), and two UGT genes ( UGT76C2, UGT83A1 ) significantly high increased by 6.74-, 10.27-, 4.98-, 10.56-, 25.67-, 16.70-, 46.92-,7.53-, 5.10-, 238.82-, 143.50-, 4.58-, 31.51-, 39.3-, 4.20-, 10.47-fold after CSA+Clo treatment compared to that in the Clo treatment. Clo down-regulated the expression of these nine GST genes and two UGT genes. These results indicated that CSA and Clo induce different kinds of metabolic enzyme-encoding genes. Similar results have been reported in previous studies. Sun et al. reported that the expression levels of ZmGSTIV , ZmGST6 , and ZmGST31 can be induced by isoxadifen-ethyl alone or in combination with nicosulfuron, but are down-regulated by nicosulfuron. The study by Zhao et al. showed that the safener isoxadifen-ethyl hydrolysate induced GSTU6 and DIMBOA UGT BX8 gene expression in rice. The metabolism of Clo appears to be influenced by five P450 genes, nine GST genes, and two UGT genes. Further investigation is required to fully understand the role of these genes in metabolizing Clo. Section title: Conclusions Educational score: 4.168875217437744 Domain: biomedical Document type: Study Language: en The findings of this study confirm that CSA can reduce the residual damage caused by Clo on maize seedlings. The soaking treatment with CSA significantly increased in chlorophyll, carotenoid and GSSG contents, reduced MDA content, and enhanced SOD and GST enzyme activities in maize seedlings compared to the Clo treatment alone. Moreover, the expression of five P450 genes ( CYP72A5 , CYP81A4, CYP81Q32, CYP81A9, CYP81A36 ), nine GST genes ( GST30 , GST31 , GSTIV , GSTVI , GST21 , GST7 , GST37, GST25 , IN2-1), and two UGT genes ( UGT76C2, UGT83A1 ) significantly high increased after CSA+Clo treatment compared to that in the Clo treatment. The pre-treatment of CSA increased the expression of 5 P450 genes, 9 GST genes, and 2 UGT genes, potentially involved in Clo metabolism in maize. The aforementioned findings provide new insights into how CSA can help reduce residual damage caused by Clo through the chlorophyll, glutathione cycle, and antioxidant enzyme activities. The expression of key genes in the glutathione metabolic pathway significantly increased under soaking with CSA. These discoveries are expected to significantly contribute to understanding the mechanism of CSA. Nevertheless, the study is subject to several constraints arising from the experimental conditions. For example, the GST enzyme activities were detected, but the potential GST-Clo metabolites could not be determined. Additionally, our study solely focused on the physiological effects of CSA on the maize shoot, without examining its impact on the root. Therefore, the findings are subject to certain limitations. Furthermore, the candidate genes identified for Clo metabolism will by functionally validated in our future work. The intention is to establish future collaborations with researchers in order to effectively address the aforementioned limitations. | Study | biomedical | en | 0.999997 |
PMC11695231 | Section title: Introduction Educational score: 4.421805381774902 Domain: biomedical Document type: Review Language: en Traumatic brain injury (TBI) refers to an acquired insult to the brain caused by an external mechanical force, which can lead to temporary or permanent impairment ( 1 ). In the United States, it is estimated that there are between 1.7 and 2.0 million cases of traumatic brain injury (TBI) occurring each year, resulting in approximately 5.3 million Americans currently experiencing long-term comorbidities linked to TBI ( 2 ). One comorbidity that is often overlooked following neurotrauma is dysregulation of the gastrointestinal (GI) organs associated with nutrient homeostasis ( 3 ). Patients suffering from gastrointestinal dysfunction frequently encounter alterations in the mucosal lining of their intestinal tract, an elevation in gut permeability, and irregularities in gut motility ( 4 , 5 ). Following brain injury, intestinal epithelial cell dysfunction or apoptosismay occur due to ischemia and/or hypoxia, oxidative stress, and inflammatory reaction. These elements can ultimately result in inflammation, ulceration, and perforation. Intestinal epithelium curtains off the contents of the lumen, preventing the invasion of pathogenic antigens. Digestive dysfunction after TBI is also associated with higher mortality and frequent hospitalizations. With the increasing number of studies on the long-term prognosis of TBI patients in recent years, more studies have proved that digestive system disorders after TBI are related to cognitive function, depression, Parkinson’s disease and other diseases after injury. However, it is still a difficult problem to clarify the molecular mechanism of digestive system dysfunction after TBI, which has a crucial role in improving the long-term prognosis of TBI patients. Section title: Epidemiology and incidence of digestive disorders post-TBI Educational score: 4.3088765144348145 Domain: biomedical Document type: Study Language: en TBI is a chronic disease process characterized by persistent secondary injury processes which can be exacerbated by subsequent challenges ( 6 ). Digestive Disorders Post-TBI can also lead to related symptoms, such as gastrointestinal bleeding ( 7 ), gastroesophageal reflux ( 8 ), and decreased intestinal motility ( 9 ). These symptoms are mainly caused by damage to the mucosa of the digestive organs and changes in their movement patterns. One study showed that gastritis was found on esophagogastroduodenoscopy in 91% of patients ( 10 ). The gut microbiota, a vast collection of microorganisms within the human digestive tract numbering in the billions, performs a multitude of physiological roles. Disruption of the gut microbiota balance plays a pivotal role in the development of numerous localized and systemic disorders ( 11 ). Consequently, the permeability of the intestinal mucosa increases. TBI-induced intestinal permeability can cause the translocation of endotoxins and bacteria in the intestinal tract, further inducing or aggravating the systemic inflammatory response and resulting in multiple organ failure and death. Section title: Types and characteristics of digestive disorders Educational score: 4.195098400115967 Domain: biomedical Document type: Study Language: en Recurrent injury to the gastroduodenal mucosa is frequently observed following a severe head trauma and manifests shortly after the incident. The major gastrointestinal changes observed after traumatic brain injury can be summarized in four aspects. Firstly, there is a reduction in blood supply to the gastrointestinal mucosa, leading to stress ulcers and bleeding in the digestive system ( 10 ). Secondly, there is a dysfunction in gut motility, resulting in symptoms such as abdominal bloating, diarrhea, delayed gastric emptying, and even intestinal paralysis ( 8 , 12 ). Thirdly, there is disruption of the gut barrier function which allows bacteria and endotoxins to enter the bloodstream and contribute to systemic inflammatory response syndrome (SIRS) and sepsis. Lastly, there are alterations in nutrient absorption by the intestinal lining which can lead to malnutrition. Section title: Assessment and diagnosis of digestive disorders in TBI patients Educational score: 4.259929656982422 Domain: biomedical Document type: Study Language: en At present, there is no unified standard for the diagnosis of digestive dysfunction after craniocerebral injury, which is mainly judged by history, clinical manifestations and related auxiliary examination. The diagnostic criteria for TBI are a clear history of trauma and confirmed as TBI by head CT or MRI. gastrointestinal injury (AGI) diagnosis criteria were evaluated according to the AGI grading system issued by the European Society of Critical Care Medicine in 2012 ( 13 ). In Zhang, D’s study, AGI score was applied to evaluate gastrointestinal functional impairment in critically ill patients ( 14 ). Although AGI score has shortcomings such as lack of objective indicators, it is still the most comprehensive method for evaluating gastrointestinal dysfunction in critically ill patients at this stage. Therefore, it can also be jointly diagnosed by laboratory and imaging examinations in clinical practice. Gastroparesis due to TBI or after nutritional support is diagnosed by bedside X-ray technique. Early identification and diagnosis of stress Cushing’s ulcer and clear source of gastrointestinal bleeding can be achieved through gastroscopy ( 15 ). Hsieh, JS ET al used gastroscopy to identify early ulcers and took endothelin-1 (ET-1), inducible nitric oxide synthase (iNOS) and macrophage inflammatory protein-1α (MIP-1α) from gastric mucosa to assess the degree of injury ( 16 ). Due to the increased intestinal permeability after TBI, harmful substances in the intestine may lead to further digestive dysfunction and even systemic inflammatory response. Hang, CH et al. Assessed intestinal barrier dysfunction by measuring the ratio of lactulose to mannitol (L/M) to determine serum endotoxin levels. Changes in fecal flora can also be used to indicate abnormal digestive system function. In the study of Nicholson, SE et al., the abundance of beneficial microbial phyla (e.g. Firmicteta) decreased when TBI patients were admitted, and opportunistic phyla (e.g. Proteobacteria) also decreased ( p < 0.05) ( 17 ). Section title: Assessment and diagnosis of digestive disorders in TBI patients Educational score: 4.061288833618164 Domain: biomedical Document type: Study Language: en The routine use of dehydrating agents such as mannitol, anesthetic sedatives, and mild hypothermia in the treatment of the majority of TBI patients inevitably heightens the risk of developing digestive disorders ( 18 ). Moreover, studies substantiate that the prolonged application of mild hypothermia not only fails to mitigate the onset of secondary brain injuries following TBI but may also trigger gastrointestinal complications including dysfunction ( 19 ). Adike, A. et al. found in their research that gastrointestinal motility disturbances represent prevalent complications among patients in critical condition, significantly contributing to mortality rates. These complications frequently correlate with sepsis, mechanical ventilation, and the administration of vasopressors, opioids, or anticholinergic medications ( 20 ). In addition, treatment measures such as mechanical ventilation and the use of vasopressors also increase the risk of gastrointestinal bleeding and intestinal dysfunction ( 21 ). Section title: Mechanisms and pathophysiology of digestive dysfunction after TBI Educational score: 3.554591655731201 Domain: biomedical Document type: Study Language: en Digestive disorders caused by TBI are mostly affected by the gut-brain Axis (GBA), a complex biphasic information exchange network between the Brain and the Gut ( 22 ). Section title: Mechanisms and pathophysiology of digestive dysfunction after TBI Educational score: 3.8110620975494385 Domain: biomedical Document type: Other Language: en The gut-brain axis signaling pathway regulates digestive system function after TBI through the interaction of vagus nerve, intestinal microbiota, immune cell response, and neuroendocrine pathways . Section title: Management and treatment approaches for TBI-related digestive disorders Educational score: 4.006726264953613 Domain: biomedical Document type: Review Language: en After TBI, due to various complex pathophysiological mechanisms, gastrointestinal motor dysfunction will lead to gastrointestinal symptoms such as vomiting, abdominal distension, diarrhea, gastric retention, and even toxic intestinal paralysis. Improving gastrointestinal motivity is the main direction of drug therapy. At present, the drugs used to improve gastrointestinal motivity mainly include metoclopramide, domperidone and Cisapride ( 37 ). Both metoclopramide and domperidone are dopamine receptor blockers, which can affect the central neuroendocrine function at the same time, but cisapride has no effect on neuroendocrine. However, with the extensive use of cisapride, problems such as prolonged Q-T interval and torsion-tip ventricular tachycardia can occur, and it is now replaced by the safer fourth-generation gastroenterokinetic drug moxapride. Komura, M., showed that mosapride reduced the incidence of gastroesophageal reflux in patients with neurologic impairment ( 38 ). In addition, there are a number of newly developed gastrointestinal motility drugs on the rise, Section title: Management and treatment approaches for TBI-related digestive disorders Educational score: 4.091858863830566 Domain: biomedical Document type: Study Language: en In recent years, Elmokadem, EM et al. proposed that the ratio of itopride to metoclopramide group could significantly increase the proportion of enteral nutritional feed, calories and protein in patients after one week ( p = 0.001), ( p = 0.002), ( p = 0.01) ( 39 ). Whether erythromycin derivative therapy enhances gastrointestinal motility remains controversial ( 40 , 41 ). In addition to gastrointestinal motivity drugs, it is also important to protect gastric mucosa, representative drugs are mainly omeprazole (OM), OM can effectively reduce the severity of stress ulcer in TBI patients by reducing the expression rate of ET-1 in tissues, iNOS and MIP-1alpha activity ( 16 ). Zhao, ZL study showed that TBI patients taking short-term high doses of glucocorticoids had an increased risk of death and a clear trend of clinical improvement compared with patients taking long-term low doses of glucocorticoids ( 42 , 43 ). Ozay, R’s study also confirmed that omeprazole and methylprednisolone were equally effective in protecting the brain from oxidative stress and early apoptosis of TBI cells ( 44 ). The study by Palm, Nicole M, et al. suggests that trauma patients at risk of stress gastropathy can discontinue preventive medications once they tolerate enteral nutrition ( 45 ), so early enteral nutrition is a top priority for TBI patients. Section title: Management and treatment approaches for TBI-related digestive disorders Educational score: 4.22170352935791 Domain: biomedical Document type: Review Language: en TBI patients are generally at risk of poor eating or disorder, high energy consumption, digestive system dysfunction, high aspiration risk, abnormal glucose and lipid metabolism, intestinal flora disorders, and poor gastrointestinal tolerance, etc. Poor nutrition management will increase the risk of poor prognosis ( 46 ). Yang, L’s study pointed out that nutritional support for TBI patients can effectively shorten the length of hospital stay, reduce the infection rate, and play a positive role in promoting the rehabilitation of TBI patients ( 47 ). Parenteral nutrition (PN) and enteral nutrition (EN) are two methods mainly used for nutritional support. Qin, Y et al. found that the use of PN was more likely to induce peptic ulcer by comparing EN and PN. Chiang, YH pointed out that compared with EN patients, the risk ratio of non-EN TBI patients was 14.63 (95% CI 8.58-24.91), which may be similar to that of EN patients ( 48 ). This may be related to the fact that EN can increase gastrointestinal blood flow, restore nitrogen balance in the body, repair intestinal mucosal barrier structure, improve gastrointestinal motivity, reduce intestinal flora and endotoxin translocation, and improve immune function ( 49 ). Therefore, in recent years, EN, especially early EN, has attracted more and more attention due to its advantages of simplicity, convenience, low economic cost, and good effect and benefit, and has become the first choice for nutritional support for TBI patients ( 50 , 51 ). Early EN refers to enteral nutrition treatment carried out within 24-48h of admission, and EEN is also recommended in ESICM Clinical Practice Guidelines published in 2017 ( 52 ). The study of Chourdakis, M showed that compared with delayed enteral nutrition, early EN can have a beneficial effect on hormone levels in patients with TBI, thereby improving prognosis ( 53 ). Section title: Management and treatment approaches for TBI-related digestive disorders Educational score: 3.9112722873687744 Domain: biomedical Document type: Review Language: en Interdisciplinary collaboration has been shown to have a positive impact on the recovery of gastrointestinal function in patients with TBI. According to a number of studies, combined intervention using multidisciplinary teams (including neurology, rehabilitation, and nutrition) can significantly improve gastrointestinal dysfunction in patients with TBI, reduce the incidence of gastrointestinal complications, and improve overall survival. The Department of Nutrition can maximize the recovery of intestinal barrier function, optimize immune function, reduce inflammatory response, and improve overall prognosis by adopting a personalized feeding regimen in enteral nutrition. Studies have shown that personalized enteral nutrition programs reduce the incidence of gastrointestinal complications and shorten the length of hospital stay in patients with TBI ( 54 ). Section title: Management and treatment approaches for TBI-related digestive disorders Educational score: 4.23625373840332 Domain: biomedical Document type: Review Language: en The addition of various nutrients to enteral nutrition is also particularly important in improving digestive function in TBI patients ( 55 , 56 ). In the study of Zhang, X, adding probiotics to enteral nutrition to treat TBI rats found that it could reduce the expression of dopamine receptor (DRs) in intestinal mucosa and prevent damage to intestinal mucosal barrier (IMB) ( 57 ). Sun, B et al. used Lactobacillus acidophilus to restore damaged Cajal interstitial cells (ICC) and damaged ICC network in TBI mice to prevent digestive disorders such as TBI-mediated inhibition of intestinal smooth muscle contractions ( 58 ). The Yi LJ study showed that supplementing EN with probiotics in early enteral nutrition effectively reduced the risk of infection, mortality, and gastrointestinal complications ( 59 ). Karakayal, EM et al. significantly improved outcomes in rats by reducing oxidative stress, apoptosis and gliosis and increasing vascular distribution through probiotic treatment ( 60 ). Cui yang et al. can induce immune tolerance by increasing Treg differentiation through oral brain protein combined with probiotics, thereby reducing secondary inflammatory injury after craniocerebral injury ( 61 ). Studies have shown that adding omega-3 fatty acids, curcumin, resveratrol, apigenin, vitamins and minerals to enteral nutrition can repair intestinal function, and ketogenic diet can improve the prognosis of patients with TBI ( 62 ). The addition of dietary fiber (DF) in enteral nutrition can produce short chainratty acids (SCFAs) under the action of human intestinal flora, thus improving digestive tract symptoms such as constipation and diarrhea in patients ( 63 ). The study of Yagmurdur, H also supports that dietary fiber should be increased while EN. Kurtz, P showed that multimodal monitoring of nutrition therapy, blood glucose control and brain microdialysis (CMD) could be used as an integrated approach to better manage TBI patients during enteral nutrition and diet adjustment. Section title: Management and treatment approaches for TBI-related digestive disorders Educational score: 4.065046787261963 Domain: biomedical Document type: Review Language: en After TBI, the brain stem, cerebellum and thalamus are damaged or intracranial high pressure leads to the inability to complete swallowing normally, resulting in swallowing disorder. In addition, dysconsciousness and cognitive decline in TBI patients may also affect swallowing function. The characteristics of traditional food do not fully meet the requirements of swallowing disorder patients, cannot cooperate with swallowing related rehabilitation training, daily meals are easy to cause coughing and aspiration. Therefore, relevant studies suggest that food traits can be changed to establish a safe and scientific diet training suitable for patients with dysphagia ( 64 ). At present, the main mainstream dysphagia training program is the International Dysphagia Diet Standardization Initiative (IDDSI), which was formulated in 2015 for thickening liquid food and daily rehabilitation training for patients with dysphagia disorders ( 65 ). Su, M et al. confirmed the feasibility of IDDSI framework in clinical and bedside applications by conducting a trial on 26 patients with dysphagia ( 66 ). An, S et al. further elaborated that the type of thickening thing seems to have more influence on the recovery of swallowing function ( 67 ). In addition to thickening liquid, some studies have pointed out that the recovery of swallowing function is also affected by the taste of sour, sweet, bitter and hot ( 68 ). Section title: Management and treatment approaches for TBI-related digestive disorders Educational score: 4.010501861572266 Domain: biomedical Document type: Study Language: en The joint rehabilitation department can improve the prognosis of patients through rehabilitation. Xing, X et al. used electroacupuncture to treat TBI patients and found that GIF, D-lactic acid (D-lac), diamine oxidase (DAO), lipolysaccharide (LPS), IAP and abdominal circumference were all lower in the acupuncture group at day 7, and compared with day 1 and day 3, The changes of the above indexes were similar (all p < 0.05). Therefore, early electroacupuncture treatment can improve digestive disorders in TBI patients. Therefore, patients with TBI need to work together with neurologists, gastroenterologists, rehabilitation doctors, dietitians, psychiatrists, etc. It is also very important for the neurosurgery department to evaluate whether early enteral nutrition can be implemented, the nutrition department to provide personalized nutritional meals, and the rehabilitation department to cooperate with the swallowing function and other rehabilitation exercise psychiatrists to pay attention to the psychological state of patients and provide necessary psychological support, so as to jointly achieve the goal of helping patients to obtain the best treatment effect and quality of life ( 48 , 69 ).Therefore, promoting collaboration among interdisciplinary teams will be an integral part of TBI therapy in the future. Section title: Prognosis and long-term outcomes of digestive disorders post-TBI Educational score: 4.242155075073242 Domain: biomedical Document type: Study Language: en The recovery of digestive system after TBI is affected by various factors, the first of which is individual differences. Weijun Fu’s study showed that female, severe GCS score, frontal injury, abnormal blood sodium, pulmonary infection, and intracranial infection are risk factors for recovery of digestive system disorders. Digestive dysfunction in people with TBI is not a short-term consequence, but rather a long-term one, and increases in incidence and severity over time ( 70 ). The majority of patients admitted to TBI for one year of rehabilitation services detected H. pylori infection. A 1-year follow-up of TBI with digestive disease found that the mortality rate was about 3 times higher than that of normal patients ( 71 ). Lower digestive tract dysfunction is a long-term and serious complication after TBI. The strong and rapid inflammatory response after TBI is not limited to the brain. With the increase of intestinal permeability, harmful substances such as intestinal bacteria enter the blood and lymphatic circulation, which can induce systemic inflammatory response and even threaten life. Mercado, NM et al., through a mouse model, found that TBI can increase systemic inflammatory response through peripheral blood albumin levels ( 72 ). Faries, PL et al. found that changes in intestinal permeability after severe trauma can further affect systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF) in patients ( 73 ). Finally, it is very likely that TBI is not a threat to patients’ lives, but the systemic inflammatory response and MODS induced by digestive disorders ( 74 , 75 ). Section title: Prognosis and long-term outcomes of digestive disorders post-TBI Educational score: 4.137931823730469 Domain: biomedical Document type: Study Language: en On the other hand, digestive disorders can aggravate the original TBI brain injury and even cause more serious chronic neurological damage ( 76 ). Therefore, with the continuous improvement of people’s requirements for quality of life, most studies not only focus on early gastrointestinal and digestive disorders after DTI, but also focus on long-term longitudinal studies ( 77 , 78 ). Celorrio, M., demonstrated in the study that antibiotic exposure 1 week after TBI reduced cortical infiltration of Ly6Chigh monocytes, increased microglial pro-inflammatory markers, and decreased T-lymphocyte infiltration, and that this effect could persist 1 month after injury. At the same time, the dysfunction of gut microbes can seriously aggravate the loss of neurons in the injured hippocampus and reduce the activation of microglia, which is accompanied by changes in the fear memory response ( 79 ), which may be related to the occurrence of mental diseases such as depression and cognitive disorders. li beixu et al. retrospectively analyzed 1027 cases of craniocerebral trauma caused by traffic accidents. The severity and area of craniocerebral trauma were closely related to the incidence of organic personality disorders ( 80 ). Studies by Chiu, LS et al. indicate that the incidence and severity of TBI is associated with an increased susceptibility to developing neurodegenerative diseases such as Parkinson’s or Alzheimer’s disease ( 81 ). So treatment requires long-term care and rehabilitation for the cognitive impairment and other clinical symptoms that people with TBI may experience. Table 1 summarizes animal experiments, clinical studies, and cohort studies on the effects of TBI on digestive system dysfunction. Section title: Current challenges and future directions in TBI-related digestive research Educational score: 3.858196973800659 Domain: biomedical Document type: Review Language: en At present, the molecular mechanism of digestive system dysfunction induced by TBI is still not fully understood. In particular, the gut-brain axis (GBA), intestinal flora and systemic inflammation have not been fully explored. In the future, by utilizing high-throughput sequencing, gene-editing techniques, immune response and metabolic pathways, as well as animal models and clinical validation, more can be revealed about the molecular mechanisms by which TBI affects digestive function. Section title: Current challenges and future directions in TBI-related digestive research Educational score: 3.6591782569885254 Domain: biomedical Document type: Review Language: en The diagnostic criteria for TBI-related digestive dysfunction are still inconsistent, which brings certain difficulties to clinical diagnosis and treatment. Most of the existing studies on TBI-related digestive dysfunction focus on the early stage after craniocerebral injury. There are still significant challenges in areas such as precision treatment strategies for TBI patients and the collection of long-term clinical data, and by filling these research gaps, we can provide more effective treatment options for TBI patients and mitigate the long-term impact of digestive complications on patient health. Section title: Current challenges and future directions in TBI-related digestive research Educational score: 4.007213592529297 Domain: biomedical Document type: Review Language: en In recent years, more and more studies have begun to focus on the neurological degeneration and psychiatric problems caused by long-term digestive disorders. Long-term follow-up data will help to better understand the natural history and prognosis of this complication. However, in Fang, Y’s study pointed out that the effective new techniques and methods required by IDDSI were not applied in time to patients with feeding difficulties after TBI ( 82 ). Therefore, cooperation is still needed in drug therapy, rehabilitation training, nutritional support and other aspects, and clinical application is still facing great challenges. Since the occurrence and presentation of digestive disorders after TBI vary from individual to individual, it is necessary to develop an individualized treatment plan for each patient’s specific situation. First, the individualized nutritional formula has more significant advantages than the conventional nutritional formula for TBI patients, which can reduce inflammation, improve the immune level of patients, improve intestinal mucosal barrier function, and have good gastrointestinal tolerance and low incidence of adverse reactions ( 83 ). Secondly, while improving the efficacy of treatment and the satisfaction of patients, individualized treatment helps to improve the self-management ability of patients, so as to participate more actively in the treatment process and better improve the prognosis ( 84 ). Section title: Current challenges and future directions in TBI-related digestive research Educational score: 4.174018859863281 Domain: biomedical Document type: Study Language: en Related studies have shown that fecal microbiota transplant-mediated Ghrelin recovery improves neural function after TBI by zhang yamei et al. ( 85 ). Berry, JAD et al. improved TBI patients’ ability to recover normal intestinal function and defecation through mesenteric elevation. Young, PJ, et al., reduced in-hospital mortality in TBI patients with selective digestive tract purification (SDD) ( 86 ). Traditional Chinese medicine treatment can also be used to effectively improve digestive disorders in TBI patients ( 87 , 88 ) Wei, C The use of ventilation Huoxuet Decoction (TQHXD) in the treatment of TBI mice showed that TQHXD could significantly improve the differentiation of cluster 36 (CD36)/15-lipoxygenase (15-LO)/nuclear receptor subfamily of 4 group A members 1 (NR4A1) signaling is expressed in mouse colon tissue to improve digestive dysfunction in TBI mice ( 89 ). Baudo, G developed in mice a Lipo-Dex (liposome nanocoliths coated with dexamethasone) that selectively targets the injured brain, thereby reducing lesion volume, cell death, astrogliosis, release of pro-inflammatory cytokines, and activation of microglia, thereby improving prognosis ( 90 ). Section title: Conclusion Educational score: 4.010436058044434 Domain: biomedical Document type: Review Language: en This paper discusses the relevant pathophysiology, diagnostic methods, and management strategies for digestive disorders following TBI. Future research is expected to further clarify diagnostic criteria and pathogenesis, particularly within the context of the GBA. This may offer new insights for clinical treatment of these disorders ( 91 , 92 ). Effective treatment requires a multidisciplinary approach to develop more personalized plans ( 35 ), with clinicians making precise diagnoses based on specific patient symptoms and signs, supplemented by laboratory tests and imaging studies. An individualized treatment plan that includes medication, nutritional support, and rehabilitation training should be developed. Early and accurate assessment of TBI patients is crucial to ensure optimal recovery and minimize complications such as digestive disorders ( 93 ). Additionally, ongoing care measures can further support the rehabilitation of TBI patients post-discharge, helping to monitor long-term gastrointestinal dysfunction and other related issues. This is vital for the comprehensive study of TBI patients ( 94 ). In summary, future collaborative, multidisciplinary research aimed at addressing this complication could reduce mortality rates in TBI patients and enhance their quality of life. | Review | biomedical | en | 0.999995 |
PMC11695233 | Section title: Introduction Educational score: 4.039257049560547 Domain: biomedical Document type: Study Language: en Ulcerative colitis (UC) is one primary form of inflammatory bowel disease (IBD), characterized by chronic inflammation of the rectum and colon, which may lead to symptoms such as rectal bleeding, increased stool frequency, decreased stool consistency, and rectal urgency with a relapsing and remitting course ( 1 ). The standard treatment for mild to moderate UC typically involves oral 5-aminosalicylic acid. In cases of moderate to severe UC, biologics such as anti-TNF agents, anti-integrins, and anti-IL-12 and IL-23 have been recommended for inducing and maintaining remission ( 2 ). Myopenia is defined as clinically relevant muscle wasting that is associated either with impaired functional capacity and/or with an increased risk of morbidity or mortality ( 3 ). Over the past decade, few studies have paid attention to the prevalence of myopenia and its correlations with clinical parameters in UC patients. Section title: Introduction Educational score: 4.03317403793335 Domain: biomedical Document type: Study Language: en The association between myopenia and prognosis has been explored in various diseases. For instance, preoperative myopenia has been identified as a negative predictive factor for cancer-specific survival and disease-free survival in patients undergoing colorectal cancer resection surgery ( 4 ). Additionally, myopenia correlates with radiographic joint damage in patients with rheumatoid arthritis ( 5 ). Moreover, in UC patients, myopenia is a risk factor for the need for surgical intervention, postoperative complications, and intravenous corticosteroid inefficacy ( 6 ). Vedolizumab, a recombinant humanized anti-α4β7-integrin monoclonal antibody, inhibits the migration of gut-homing memory T cells into the gastrointestinal submucosa, thereby reducing intestinal inflammation specifically ( 7 ). Vedolizumab has shown effectiveness in both inducing and maintaining remission in UC patients and has been increasingly used in the treatment of UC ( 8 ). However, data regarding the impact of muscle mass on response to vedolizumab in UC patients was sparse. Section title: Introduction Educational score: 4.024265766143799 Domain: biomedical Document type: Study Language: en In addition to skeletal muscle, adipose tissue is another important component of body composition. It also gained some attention on its implications in disease prognosis, though less than muscle. Previous studies primarily focused on subcutaneous and visceral fat, with the latter predominantly comprising mesenteric and omental adipose tissues ( 9 ). Visceral adipose tissue can induce insulin resistance through proinflammatory cytokine and adipokine secretion, contributing to metabolic disorders ( 10 ). Subcutaneous adipose tissue depletion accelerates cachexia in cancer patients and leads to poor outcomes ( 11 ). Higher ratio of visceral fat area to subcutaneous fat area has been reported to be associated with higher possibility of undergoing surgery and higher frequency of disease flare in IBD patients ( 12 , 13 ). However, adipose tissue distribution in UC patients and its correlation with therapeutic outcomes remain unclear. Section title: Introduction Educational score: 4.089432716369629 Domain: biomedical Document type: Study Language: en This study aimed to investigate muscle mass loss and adipose tissue distribution in a cohort of patients with active UC, analyze the relationship between body composition and clinical data, and explore associations between body composition and response to vedolizumab therapy. Section title: Participants Educational score: 4.083126068115234 Domain: biomedical Document type: Study Language: en Hospitalized patients aged 18 to 70 with active UC at Peking Union Medical College Hospital (PUMCH) were consecutively enrolled from November 2014 to October 2022. Inclusion criteria involved: (1) with complete medical records; (2) undergoing Computed tomography (CT) scans including the third lumbar vertebrae (L3) cross-section within a week before or after admission; (3) no UC surgery history; (4) had serum albumin (ALB) and high-sensitivity C-reactive protein (hsCRP) tests within a week before or after the CT examination mentioned above. Exclusions comprised comorbidities in major organs or auto-immune diseases beyond UC, spondyloarthropathy-related manifestations, neuromuscular or orthopedic issues impacting muscle health, cancer history, non-UC admissions, and pregnant or lactating females. For patients readmitted for UC recurrence or exacerbation, screening for inclusion/exclusion criteria was based on their initial admission. The study was approved by the Ethics Committee of PUMCH (No. I-22PJ700) and was conducted following the declaration of Helsinki. Section title: Measurement of body composition by computed tomography Educational score: 4.1274495124816895 Domain: biomedical Document type: Study Language: en The CT data were obtained using the Picture Archiving and Communication System. The L3 muscle region has been demonstrated to best predict overall body composition ( 14 ). Skeletal muscle area (SMA), visceral fat area (VFA), and subcutaneous fat area (SFA) were quantified on L3 CT images using Syngo.via software (Siemens Healthineers, Forchheim, Germany) with specific Hounsfield Unit (HU) ranges for muscles (−29 to +150), subcutaneous adipose tissue (−190 to −30), and visceral adipose tissue (−150 to −50) ( 15 ) . SMA, VFA, and SFA values were normalized to height squared (m 2 ), yielding skeletal muscle index (SMI), visceral fat index (VFI), and subcutaneous fat index (SFI) in cm 2 /m 2 , respectively. Myopenia was defined as SMI < 44.77 cm 2 /m 2 in males and <32.50 cm 2 /m 2 in females based on extensive research in China ( 16 ). Additionally, mean HU for L3 muscle assessed muscle quality, with lower values indicating greater fat infiltration, namely, myosteatosis ( 17 ). The VFA/SFA ratio (VSR) reflected the extent of visceral obesity. Section title: Data collection Educational score: 4.077503204345703 Domain: biomedical Document type: Study Language: en Demographic and clinical data including age, sex, body mass index (BMI), non-volitional weight loss, the percentage of food intake reduction, and disease duration were obtained from medical records. Disease activity was assessed following the modified Truelove and Witts’ criteria. Nutritional risk was evaluated using the Nutritional Risk Screening 2002 , while malnutrition was diagnosed based on the Global Leadership Initiative on Malnutrition (GLIM) guidelines ( 18 , 19 ). Results of serum ALB, hsCRP, and serum lipid tests [including triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C)] within a week before or after the CT examination were collected. Section title: Exploration of the correlation between myopenia and clinical response to vedolizumab Educational score: 4.053985595703125 Domain: biomedical Document type: Study Language: en Patients treated with vedolizumab for induction and maintenance therapy over a minimum of 6 months, with CT scans containing L3 taken within 3 months before vedolizumab initiation, were included. The vedolizumab regimen consisted of 300 mg intravenously at weeks 0, 2, and 6, followed by doses every 8 weeks. Clinical response to vedolizumab was evaluated using the Mayo score for UC until the last vedolizumab administration before September 2023, with a positive response defined as a ≥3-point reduction in the Mayo score compared to baseline pre-vedolizumab ( 20 ). Section title: Statistical analyses Educational score: 3.9733285903930664 Domain: biomedical Document type: Study Language: en Statistical analyses were performed using SPSS version 23.0 (SPSS Inc., Chicago, IL, United States) and GraphPad Prism version 6.0 (GraphPad Software Inc., San Diego, CA, United States). Continuous variables were expressed as mean ± standard deviation (SD) for normal distributions or median (Q1, Q3) for non-normally distributed data. Categorical variables were presented as numeric values (percentages). Group comparisons for continuous variables were performed using independent samples t -tests for normal distributions and the Mann–Whitney U -test for non-normal distributions. Categorical variable comparisons utilized the Chi-squared test, with significance set at p < 0.05. Pearson’s correlation or Spearman’s correlation was used to analyze associations between parameters based on the distribution characteristics of data. Multivariate logistic regression analysis identified risk factors for vedolizumab non-response, preceded by variance inflation factor calculations to check for collinearity among covariates (collinearity considered if variance inflation factor > 5). Section title: Prevalence of myopenia in UC patients Educational score: 4.074228286743164 Domain: biomedical Document type: Study Language: en A total of 457 hospitalized patients with active UC were analyzed, including 254 males and 203 females . Demographic, clinical, muscle, and fat indices data from CT scans are presented in Table 1 . No significant differences were observed in age or disease duration between myopenic and non-myopenic patients. Myopenia prevalence was 49.7% (227 patients) in the overall cohort, with a slightly but not significantly higher rate in males (52.0%) than in females (46.8%). Myopenic patients had significantly lower VFA, VFI, SFA, and SFI values but a higher VSR than non-myopenic patients, denoting distinct fat distribution patterns in the two populations. Additionally, mean HU and ALB levels were significantly lower, while hsCRP levels were significantly higher in the myopenia group. Section title: Prevalence of myopenia in UC patients Educational score: 4.09089994430542 Domain: biomedical Document type: Study Language: en The prevalence of myopenia increased with disease activity, with proportions of 34.3, 43.7, and 62.7% in patients with mild, moderate, and severe disease activity, respectively. Myopenia was differently distributed between younger and older patients. In patients aged 18–50 years, the prevalence of myopenia was 44.9%, while in patients over 50 years, the prevalence of myopenia was 64.3% ( p < 0.001). Among patients with BMI < 18.5 kg/m 2 and ≥18.5 kg/m 2 , myopenia rates were 75.0 and 39.4%, respectively. Notably, within the myopenia subset, 56.4% had a BMI ≥ 18.5 kg/m 2 , while 4.0% had a BMI ≥ 24.0 kg/m 2 , with only one male patient surpassing a BMI of 28.0 kg/m 2 . Section title: Association between myopenia and malnutrition Educational score: 4.094130992889404 Domain: biomedical Document type: Study Language: en Among all the patients, 77.7% (355/457) were at nutritional risk according to NRS-2002, with 57.7% (205/355) of them exhibiting myopenia. Based on GLIM criteria, malnutrition prevalence was 67.2% (307/457). The rate of malnutrition was significantly higher in UC patients with myopenia compared with those without myopenia (86.8% vs. 47.8%, p ≤ 0.001, Table 1 ). No significant sex-based disparity was observed between malnourished and well-nourished groups ( Table 2 ). Section title: Differences in body composition between male and female UC patients Educational score: 4.084072589874268 Domain: biomedical Document type: Study Language: en The age, disease duration, disease activity, and serum hsCRP showed no significant difference between male and female patients. SFI was significantly higher in female patients, while BMI, VFI, VFR, and mean HU were all significantly lower in female patients ( Table 2 ). Irrespective of sex, mean HU showed significantly negative correlations with SFI ( r = −0.179, p = 0.011 in females, and r = −0.289, p < 0.001 in males), VFI ( r = −0.443, p < 0.001 in females, and r = −0.510, p < 0.001 in males), and VSR ( r = −0.421, p < 0.001 in females, and r = −0.508, p < 0.001 in males), especially with VFI and VSR. Section title: Correlations between muscle or fat indices and laboratory indices in UC patients Educational score: 4.095062255859375 Domain: biomedical Document type: Study Language: en The SMI and mean HU showed significantly positive associations with ALB while VSR showed significantly negative associations with ALB, regardless of sex . SFI showed a significantly positive association with ALB only in female patients ( r = 0.233, p = 0.001). Among the 222 patients with ALB levels <35 g/L, which is the cut-off value for hypoalbuminemia, 63.5% (141/222) had myopenia. Among the 235 patients with ALB levels ≥35 g/L, the prevalence of myopenia remained high at 36.6% (86/235). Section title: Correlations between muscle or fat indices and laboratory indices in UC patients Educational score: 4.0587158203125 Domain: biomedical Document type: Study Language: en Conversely, SMI and mean HU were negatively associated with hsCRP, and VSR showed a significantly positive association with hsCRP in female patients and a tendency of positive association with hsCRP in male patients ( Table 3 ). Interestingly, hsCRP only showed a weak negative association with BMI in male patients ( r = −0.129, p = 0.039) and showed no significant association with BMI in female patients. Section title: Correlations between muscle or fat indices and laboratory indices in UC patients Educational score: 4.079299449920654 Domain: biomedical Document type: Study Language: en Subsequently, we explored the association between serum lipid levels and fat indices. Of all patients, 312 patients underwent serum lipid tests including TG, TC, and LDL-C within 1 week before or after CT. SFI showed a significantly positive association with TG. Moreover, a significant positive association between VFI and TG was observed ( Table 4 ). There were positive associations between SFI or VFI with TC or LDL-C as well, and the associations with LDL-C were stronger than those with TC ( Table 4 ). However, there were no significant associations found between mean HU or VSR with TG, TC, or LDL-C (data not shown). Section title: Correlations between body composition and clinical response to vedolizumab in UC patients Educational score: 4.1875410079956055 Domain: biomedical Document type: Study Language: en In our cohort, there were 92 patients (42 females and 50 males) who received vedolizumab and had CT images containing L3 cross-section within 3 months before the initiation of vedolizumab therapy. There were 67 patients with moderate disease activity and 25 patients with severe disease activity. The median therapy duration was 18 months, with an overall clinical response rate of 70.7% (65/92). When dividing these patients according to muscle mass, 36 patients had myopenia and 56 patients did not. The clinical response rate was significantly lower in the myopenia group [52.8% (19/36) vs. 82.1% (46/56), p = 0.003]. However, when comparing patients with a BMI < 18.5 kg/m 2 to those with a BMI ≥ 18.5 kg/m 2 , no significant difference was observed in the clinical response rates (68.2% vs. 71.4%). Visceral obesity, defined as VSR ≥ 75th centile of sex-specific VSR in Table 2 , was observed in 25 patients, and the clinical response rate showed a tendency to be lower in patients with visceral obesity but did not reach significance (60.0% vs. 74.6%, p = 0.170). Myosteatosis, defined as mean HU ≤ 25th centile of sex-specific mean HU in Table 2 , was observed in 15 patients, with no significant difference observed in the clinical response rates between patients with and without myosteatosis. Section title: Correlations between body composition and clinical response to vedolizumab in UC patients Educational score: 4.119794845581055 Domain: biomedical Document type: Study Language: en Univariate analysis was performed on the baseline characteristics of the response group and non-response group ( Table 5 ). The proportions of patients with malnutrition did not differ significantly between groups. Both groups showed no significant differences in age and sex distribution. Except for SMI, patients with and without clinical response to vedolizumab showed no significant difference in BMI or any of the other muscle and fat indices. Then, multivariate logistic regression showed that myopenia remained significantly associated with non-response to vedolizumab after adjusting for vedolizumab treatment duration, ALB, and hsCRP (OR = 3.458, 95% CI 1.238–9.659, p = 0.018). Section title: Discussion Educational score: 4.107076644897461 Domain: biomedical Document type: Study Language: en This study provided some significant insights. The prevalence of myopenia was 49.7% among hospitalized patients with active UC, escalating to 64.3% in individuals aged over 50 years. Notably, 67.2% of patients were diagnosed with malnutrition according to GLIM criteria, close to our previous report ( 19 ). Among those with malnutrition, 64.2% had myopenia. This study is pioneering in its focus on sex variations in body composition based on CT scans in UC patients. It focuses on the relationship between body composition and clinical data in females and males, respectively. The indices reflecting muscle quantity and quality consistently showed positive associations with ALB levels and negative associations with hsCRP levels, whereas the parameter reflecting visceral fat accumulation showed an opposing trend. Moreover, this study described the relationship between baseline body composition and the clinical response to vedolizumab in UC patients, revealing myopenia as a potential predictor for poor response to vedolizumab. Section title: Discussion Educational score: 4.133268356323242 Domain: biomedical Document type: Study Language: en Similar to our results, a recent single-center study involving 173 UC patients reported a myopenia prevalence of 53.2% ( 21 ). Zhang et al. ( 22 ) reported that the prevalence of myopenia in UC patients was 27.3%, but the sample size was relatively small ( n = 99). The prevalence of myopenia spiked to over 60% in UC patients with severe disease activity in our cohort, consistent with reports indicating myopenia rates ranging from 50.2 to 69.5% among patients with acute severe UC ( 23 , 24 ). Notably, over 50% of UC patients with myopenia had normal BMI levels in our cohort, consistent with earlier studies indicating that 51.2–60.1% of IBD patients with myopenia had a BMI ≥ 18.5 kg/m 2 ( 25 , 26 ). Furthermore, we found that only 4.0% of overweight or obese UC patients had myopenia, highlighting a lesser likelihood of myopenia occurrence in such patients despite probable weight loss during the disease course, whereas some previous studies reported that the rates could be as high as 12.6–19.5% ( 25 , 26 ). Section title: Discussion Educational score: 4.1202778816223145 Domain: biomedical Document type: Study Language: en Although muscle mass has a crucial role in diagnosing malnutrition ( 18 , 27 ), it is essential to note that low muscle mass does not always equate to malnutrition ( 28 ) since identifying nutritional risk stands as the primary step in diagnosing malnutrition, while our results showed that 42.3% of UC patients at nutritional risk had normal muscle mass. The relationship between muscle mass loss and malnutrition in UC patients has been sparsely explored. Given that nearly 40% of UC patients with a BMI ≥ 18.5 kg/m 2 manifested myopenia on CT scans, evaluating muscle mass in UC patients is recommended to avoid overlooking those with poor nutritional status which can be hidden by a normal BMI. Our analysis showed that SMI had a stronger association with hsCRP compared to BMI in UC patients, hinting at inflammation’s greater impact on lean body mass loss than on body weight decline. Moreover, we found that myopenia, rather than low BMI before vedolizumab initiation, might increase the risk of treatment failure. A recent study on the association between low muscle mass and colectomy in acute severe UC patients also underscored myopenia, not low BMI, as a risk factor for rescue therapy and colectomy ( 26 ). Taken together, maintaining healthy muscle condition in UC patients may hold a stronger connection with disease severity and therapeutic effectiveness than normal weight, warranting increased attention in the management of UC in the future. Gastroenterologists and nutritionists can choose the measurement approach of muscle mass within their reach, like bioelectrical impedance analysis, Dual-energy X-ray absorptiometry, CT, and MRI. Section title: Discussion Educational score: 4.471441268920898 Domain: biomedical Document type: Study Language: en Skeletal muscle, constituting about 40% of total body weight, represents the largest tissue in the human body ( 29 ). It undergoes dynamic changes due to factors like aging, illness, reduced physical activity, poor nutrition, and specific medications ( 3 ). Mitchell et al. reported a median muscle mass loss rate of 4.7% per decade in men and 3.7% per decade in women ( 30 ). As expected, we observed a remarkably higher prevalence of myopenia in UC patients over 50 years old compared to those aged between 18 and 50 years. However, it is noteworthy that in contrast with “inflammaging,” a term describing low-grade chronic systemic inflammation associated with aging ( 31 ), disease-induced inflammation can be more potent, serving as a primary trigger for rapid muscle mass depletion even in young patients ( 32 , 33 ). Originating from colonic inflammatory conditions, UC triggers an upsurge in pro-inflammatory cytokines such as interleukins, TNF- α , and TGF- β ( 34 ). This systemic inflammation can inhibit the IGF-1/mTORC1 pathway, leading to increased protein catabolism and decreased muscle protein synthesis, recognized as a key mechanism for muscle wasting in UC ( 35 , 36 ). Moreover, hypovitaminosis D, which is common in UC patients ( 37 ), deserves more attention in both systematic inflammation and myopenia in UC. Decreased 1,25-dihydroxycholecalciferol [1,25(OH)2D] and upregulated IL-33/IL-31 axis can alter the balance between inflammatory Th1/Th17 cells and T regulatory (Treg) cells and promote the bacterial translocation, associated with the pathophysiological processes of autoimmune diseases like IBD ( 38 , 39 ). On the other hand, vitamin D insufficiency is correlated with reduced muscle function and sarcopenia ( 40 ). Meanwhile, skeletal muscle is acknowledged as a secretory organ capable of releasing myokines that counteract the detrimental effects of pro-inflammatory cytokines and alleviate inflammation ( 41 ). Our findings revealed a significant negative correlation between L3-SMI and serum hsCRP in UC patients, emphasizing the intricate but non-causal interaction between skeletal muscle and systematic inflammation. Section title: Discussion Educational score: 4.239439964294434 Domain: biomedical Document type: Study Language: en In this study, we comprehensively investigated the links between subcutaneous fat, visceral fat, intramuscular fat, and systemic inflammation in active UC patients. We observed the positive associations between myosteatosis/visceral obesity and systematic inflammation in UC patients, as evinced by the negative correlation between mean HU and serum hsCRP as well as the positive correlation between VSR and serum hsCRP. Visceral and intramuscular fats have been shown to secrete an abundance of proinflammatory cytokines and cause the accumulation of proinflammatory immune cells ( 9 , 42 ). Conversely, subcutaneous fat appears to have beneficial effects, evidenced by improved outcomes in gastric cancer patients with higher SFA and CD patients with increased SFI at the L3 level ( 43 , 44 ). While we found no significant correlation between SFI and hsCRP, a positive correlation between SFI and ALB in female UC patients was observed in this cohort. Aligning with observations in healthy adults ( 45 ), our results demonstrated that both VFI and SFI positively correlated with serum TG, TC and LDL-C. Our team’s descriptive review proposed that blood lipids could be pivotal in fat redistribution, influencing the formation of visceral fat depots and fat infiltration in muscles and organs ( 42 ). The close relationship we noted between serum lipids and visceral fat bolsters this perspective. Interestingly, the higher serum TG level has been demonstrated to be associated with a higher possibility of surgery in UC patients ( 46 , 47 ), which might be partly explained by the intimate connections among blood lipid level, body fat distribution, and systematic inflammation. Therefore, visceral and intramuscular fat may provide prognostic value to some degree in UC patients if they could be assessed regularly. Section title: Discussion Educational score: 4.187668800354004 Domain: biomedical Document type: Study Language: en Similar to patients with cancer cachexia ( 48 ), we observed that UC patients with reduced muscle mass also had reduced subcutaneous fat and visceral fat. Systemic inflammation not only plays an crucial part in the waste of muscle and adipose tissue in UC patients, but also is of central importance in the mechanism of cachexia development ( 49 ). However, unlike in myopenic UC patients, the increase of serum hsCRP is not common in patients with cancer cachexia ( 50 ). Despite the reduction of both visceral and subcutaneous fat in myopenic UC patients, we found a higher VSR in this group. Considering higher systematic inflammation level (indicated by higher serum hsCRP) in myopenic UC patients, the stronger link between visceral fat and systemic inflammation than subcutaneous fat may be the critical factor leading to less waste of visceral fat than subcutaneous fat ( 9 ). Several studies have investigated the application of interventions that may improve muscle mass or muscle function in IBD patients. A study with a small sample size ( n = 20) showed that 8-week moderate-intensity combined aerobic and resistance training could increase lean tissue mass and decrease fat mass in IBD patients ( 51 ). Moreover, 4-week resistance training together with whey protein was reported to significantly increase muscle mass in IBD patients when compared with resistance training and placebo ( 52 ). Collectively, structured exercise can decrease visceral fat mass and increase muscle mass, which has the potential to be an adjunctive therapy in IBD management ( 53 ). Section title: Discussion Educational score: 4.27005672454834 Domain: biomedical Document type: Study Language: en A recently published article including 95 IBD patients reported that the total rate of clinical improvement after using vedolizumab was 74.8%, with an average duration of 17.83 months ( 54 ). Similarly, our cohort of UC patients treated with vedolizumab had a clinical response rate of 70.7% over a median duration of 18 months. Muscle loss can instigate a pro-inflammatory milieu due to deficient myokine signaling and compromised regenerative capabilities of immune cells ( 55 ), potentially leading to immunotherapy resistance. Therefore, better maintenance of skeletal muscle homeostasis may result in healthier immune functionality and a higher response rate to biologics. Recently, the relationship between visceral obesity and biologics treatment response in IBD patients has been explored in a few studies. A study including 68 IBD patients (with only 5 UC patients) reported that VSR was not correlated with treatment failure of anti-TNFα ( 56 ), while another study including 99 patients with Crohn’s disease (CD) found that a high VFI:SMI ratio was associated with an increased risk of failing standard doses of ustekinumab ( 57 ). A recently published study investigated the correlation between different biologics and endoscopic remission in IBD patient, including 141 patients in total (79 CD patients and 62 UC patients, with 52 patients using infliximab, 43 patients using ustekinumab, and 46 patients using vedolizumab), showing that patients (whether CD or UC patients) with higher visceral fat level were less likely to achieve endoscopic remission after the biologics treatment ( 58 ). In this study, we noted a lower albeit statistically insignificant clinical response rate in patients with visceral obesity, suggesting a potential adverse role of visceral obesity in inflammation regulation by vedolizumab in UC patients. Considering the important position of biologics in the therapy of IBD, the role of visceral obesity in the biologics response in IBD patients deserves further attention in the future. Section title: Discussion Educational score: 4.066156387329102 Domain: biomedical Document type: Study Language: en There are several limitations in this study. Firstly, while CT scans offer an approach to evaluate muscle mass retrospectively, muscle strength and physical performance could not be assessed, precluding us from making the diagnosis of sarcopenia in these individuals. Secondly, it should be noted that UC patients who need to undergo CT examinations may have more severe disease manifestations than those who do not. Given that myopenia prevalence rises with disease severity, as exhibited in this study, the prevalence of myopenia might be overestimated. Similarly, in UC patients with milder disease activity (such as patients in Gastroenterology clinics), a lower prevalence of myopenia can be expected compared to the data in our cohort. Therefore, multi-center studies may help to enhance the representativeness of myopenia prevalence in hospitalized UC patients. Additionally, the results of the preliminary exploration of the relationship between body composition and vedolizumab response are incapable to determine causality, and need to be verified in a larger cohort in the future. Section title: Conclusion Educational score: 4.073356628417969 Domain: biomedical Document type: Study Language: en This study demonstrated that almost 50% of hospitalized patients with active UC had myopenia. Muscle quantity and quality at L3 showed a significant positive correlation with ALB and a negative correlation with hsCRP, while the degree of visceral obesity displayed an opposite pattern. Myopenia showed significant association with poor clinical response to vedolizumab. The assessment and optimization of body composition should receive more consideration in the future management of UC. | Study | biomedical | en | 0.999996 |
PMC11695235 | Section title: Introduction Educational score: 4.272001266479492 Domain: biomedical Document type: Review Language: en The endoplasmic reticulum (ER) is a membrane-bound organelle that plays a central role in the synthesis, folding, and transport of proteins, as well as lipid production and calcium storage. It is a dynamic structure, adapting and remodeling itself in response to cellular demands. This adaptability is critical for maintaining cellular proteostasis, thereby balancing protein synthesis, folding, and degradation. ER stress occurs when the capacity of the ER to properly fold proteins is overwhelmed, leading to an accumulation of misfolded or unfolded proteins in the ER. ER stress triggers the activation of cellular signaling pathways designed to take corrective actions and restore proteostasis, or to initiate cell death pathways if the burden is prolonged or severe. While ER stress and downstream signaling can occur in any cell, those with a high demand for protein synthesis such as hormone-producing cells are particularly susceptible. The placenta is one such organ tasked with producing numerous hormones and other proteins critical for fetal development and pregnancy success, where disruptions in proteostasis can have severe consequences for maternal and fetal health. In this review, we will present the importance of ER homeostasis for placental development and function, and discuss evidence linking ER stress with deficient placentation and adverse pregnancy outcomes. Section title: The placenta Educational score: 4.377654075622559 Domain: biomedical Document type: Review Language: en The placenta is a transient yet highly sophisticated organ that intimately connects the gestational parent and fetus. It forms the primary interface separating maternal and fetal tissue and facilitates metabolic exchange, endocrine functions, and fetal protection. The placenta regulates the exchange of nutrients, oxygen, carbon dioxide, and other substances between maternal and fetal circulations, while restricting the transfer of pathogens, xenobiotics, maternal immune cells and many other potentially harmful substances from accessing fetal blood ( 1 , 2 ). The placenta is also an adaptive organ that can integrate information on maternal nutrient availability and fetal demands, and undergo dynamic morphological and molecular changes to support fetal development ( 3 ). As an endocrine organ, the placenta produces a plethora of hormones and other factors that regulate maternal adaptations to pregnancy along with fetal growth and development ( 4 , 5 ). Section title: An overview of placental structure Educational score: 4.7238383293151855 Domain: biomedical Document type: Study Language: en The human placenta is arranged into highly branched tree-like villi containing an inner core of macrophages, fibroblasts, pericytes, connective tissue, and capillaries that connect to the fetal circulation through the umbilical vessels. The villous core is separated from maternal blood by a trophoblast bilayer: an inner portion of cytotrophoblasts (CTBs) resting on a basement membrane, and an outer syncytiotrophoblast (STB) layer . CTBs are self-renewing progenitor cells that initially form a continuous layer during early pregnancy, and replenish the STB layer by fusing into it. In late pregnancy, the proportion of CTBs dwindles and they form a disjointed layer beneath the STB. The STB layer, on the other hand, is a multinucleated entity with a vast interconnected cytoplasm that lines the placental villi and bathes in maternal blood. It forms the primary boundary between maternal blood and fetal tissue, and is responsible for numerous placental functions including maternal-fetal gas and nutrient exchange ( 6 , 7 ). Given its proximity to maternal blood, STB is responsible for the production and metabolism of numerous hormones, such as human chorionic gonadotropin (hCG). These factors are deposited into maternal circulation to alter maternal physiology and metabolism for fetal benefit. Furthermore, since the STB does not contain intercellular junctions, it forms a semi-exclusive barrier that restricts the passage of many substances from accessing fetal circulation ( 8 ). Section title: An overview of placental structure Educational score: 4.386212348937988 Domain: biomedical Document type: Study Language: en At sites where large anchoring villi contact the decidua basalis (the specialized endometrial tissue adjacent to where the placenta forms), CTBs differentiate into a distinct cell-type: extravillous trophoblasts (EVTs). Proximally, EVTs proliferate into stratified cellular columns. At the distal tips of these columns, cells stop replicating and gain invasive properties. Invasive EVTs infiltrate into the decidua basalis and inner third of the myometrium. EVTs are versatile cells that affix the placenta to the decidua basalis, interact with decidual stromal cells and immune cells to support immunological tolerance, and remodel uterine blood vessels and glands to ensure that a consistent supply of nutrients and oxygen are delivered to the placenta to support fetal sustenance ( 9 ). Section title: An overview of placental structure Educational score: 4.379774570465088 Domain: biomedical Document type: Study Language: en The decidua basalis is a dynamic tissue derived from the endometrium adjacent to where the placenta forms. Transformation of the endometrium into the decidua begins during the secretory phase of the menstrual cycle and accelerates upon fertilization. Decidualization involves the differentiation of fibroblast-like endometrial stromal cells (ESCs) into polygonal decidual stromal cells (DSCs) along with extensive development of uterine glands and blood vessels, processes that are tightly regulated by estrogen and progesterone ( 10 ). Transformation of ESCs into DSCs is associated with a substantial increase in the metabolic demand and secretory activity of the cells that serve essential roles in early embryo nutrition and maternal-fetal communication ( 11 ). Section title: An overview of placental structure Educational score: 4.214247703552246 Domain: biomedical Document type: Review Language: en Placental structure exhibits marked diversity between species. In humans and closely-related primates, the placenta is termed “hemochorial” since maternal blood directly contacts trophoblasts. Other species with hemochorial placentation include common laboratory rodents like mice, rats, and guinea pigs. Although these species exhibit notable differences in placental anatomy and physiology compared to humans, there are also many similarities. For example, placentas of mice, rats, and guinea pigs have a labyrinth zone containing syncytialized trophoblasts that specialize in nutrient and gas exchange (akin to the placental villi in humans), and a junctional zone adjacent to the decidua basalis that anchors the placenta to the decidua and is the site where invasive trophoblasts emanate (analogous to human EVTs). Consequently, rodents are often used as laboratory models to gain deeper mechanistic insight into placental development and maternal-placental-fetal interactions ( 12 ). Although this review will focus mostly on placentation in humans, much knowledge has been derived from studies using rodent models and these studies will be mentioned when appropriate. Section title: Placenta-associated pregnancy complications Educational score: 4.124724388122559 Domain: biomedical Document type: Review Language: en Deficient placental development and function is a major culprit underlying severe pregnancy complications that compromise maternal and fetal well-being and survival. For example, inadequate placental development resulting in placental insufficiency (inadequate maternal blood supply to the placenta) can lead to preeclampsia—a common and dangerous pregnancy disorder characterized by sudden-onset maternal hypertension, endothelial dysfunction, and organ damage. In many cases, in preeclampsia the fetus does not obtain adequate oxygen and nutrients, resulting in fetal growth restriction (FGR) and the potential for long-term health deficiencies. Placentas from preeclampsia and FGR often exhibit evidence of hypoxia, oxidative stress and inflammation—conditions that can adversely affect ER homeostasis and result in ER stress ( 13 – 15 ). In this review, we will discuss ER homeostatic mechanisms required for placental development and function, and describe how these mechanisms are perturbed following cellular exposure to stress during placenta-associated pregnancy complications. Section title: ER stress Educational score: 4.246695041656494 Domain: biomedical Document type: Study Language: en Protein turnover in the placenta increases throughout pregnancy, requiring efficient quality control mechanisms to ensure that the protein folding capacity of the ER meets protein synthesis demands ( 16 , 17 ). Key components of this quality control machinery include ER-resident chaperones such as binding immunoglobulin protein (BiP; also called glucose regulated protein 78, GRP78), calreticulin, calnexin, and protein disulfide isomerases (PDIs). These chaperones assist proteins in achieving their native conformation ( 18 ). However, if chaperones cannot keep pace with protein folding demands, an accumulation of unfolded or misfolded proteins in the ER lumen can result, leading to ER stress and the activation of the unfolded protein response (UPR) ( 19 ). The role of ER-associated chaperones for placentation and decidualization are summarized in Supplementary Table 1 . Section title: ER stress Educational score: 4.51422643661499 Domain: biomedical Document type: Study Language: en ER stress can occur due to an accumulation of abnormal lipids or misfolded proteins in the ER ( 20 , 21 ). The accumulation and aggregation of misfolded proteins can occur due to increased rates of protein synthesis, deficiency in autophagy, nutrient deprivation, and dysregulated calcium levels ( 22 , 23 ). Many cell types experience a high protein load during various stages of differentiation and maturation, resulting in ER stress. Induction of ER stress can have morphological and biochemical consequences to cells. Morphologically, ER stress can result in dilation of ER cisternae to increase the lumen size and accommodate the sudden increase in protein accumulation ( 24 , 25 ). Many cells that experience ER stress undergo a degree of epithelial-to-mesenchymal transition, including increased N-cadherin levels and decreased E-cadherin levels ( 26 ). Biochemically, ER stress can activate several signaling pathways including the UPR, in an effort to resolve the stress and restore proteostasis ( 27 ). Section title: ER stress Educational score: 4.581161022186279 Domain: biomedical Document type: Study Language: en Recent studies have characterized early-onset preeclampsia as a proteinopathy due to presence of misfolded proteins in maternal urine and serum ( 28 – 30 ). Misfolded oligomeric proteins and amyloids, arising from defective chaperone function or impaired autophagy, may either deposit in the placenta from maternal circulation or be produced directly by placental cells ( 31 ). Several amyloidogenic proteins, including amyloid-β, transthyretin, and α-1-antitrypsin, have been identified in the preeclampsia-associated misfoldome and proposed as potential diagnostic markers for preeclampsia ( 31 – 33 ). Deposition of protein aggregates in the placenta may induce cellular toxicity, impair nutrient and gas exchange, and lead to placental insufficiency. Exposure of placental explants to serum from preeclamptic pregnancies induces ER stress and the formation of syncytial knots, suggesting that maternal serum in preeclampsia may contain ER stress-inducing factors that promote protein aggregation in placental cells ( 34 , 35 ). While the placenta can efflux misfolded proteins into maternal blood, the accumulation of protein aggregates in maternal blood may pose risks for maternal organs ( 31 ). Therefore, targeting of misfolded proteins and resolution of ER stress could be promising therapeutic strategies to restore cellular homeostasis and improve pregnancy outcomes ( 36 – 38 ). Section title: The UPR in placentation Educational score: 4.299806594848633 Domain: biomedical Document type: Review Language: en The UPR is a conserved cellular response to ER stress. In principle, the UPR seeks to correct the accumulation of unfolded and misfolded proteins in the ER, thereby restoring ER homeostasis. Basal levels of UPR activation promote cellular homeostasis, but in cases of prolonged or severe ER stress, unmitigated UPR activation can lead to cell death ( 39 , 40 ). The UPR pathway consists of three ER-resident sensor proteins: inositol-requiring enzyme 1 (lRE1), protein kinase R-like ER kinase (PERK), and activating transcription factor 6 (ATF6), all of which trigger a transcriptional response to adapt the ER folding environment by upregulating genes involved in protein folding and degradation ( 41 ). The following sections will summarize the role of IRE1, PERK, and ATF6 signaling in the context of placentation and decidualization. Section title: IRE1 Educational score: 4.992242813110352 Domain: biomedical Document type: Study Language: en IRE1 is a transmembrane protein that is evolutionarily conserved from yeast to humans ( 42 , 43 ). It contains a serine/threonine kinase domain and a cytosol-facing domain with endoribonuclease activity. There are two isoforms of IRE1 in mammals – IRE1α and IRE1β – with IRE1α being the ubiquitously expressed isoform ( 44 ). Upon detection of misfolded or unfolded proteins, IRE1 homodimerizes and activates its kinase function through trans-autophosphorylation of Ser 724 , Ser 726 , and Ser 729 in the kinase activation loop ( 45 ). Once activated, IRE1 splices out a 26-nucleotide intron from the X-box binding protein 1 ( XBP1 ) mRNA sequence, encoding for the spliced XBP1 (XBP1s) transcription factor ( 46 ). Thus, IRE1 activation is typically measured through the phosphorylation of IRE1, splicing of XBP1 mRNA, or the accumulation and nuclear localization of the XBP1s transcription factor. XBP1s transcriptionally regulates genes encoding various chaperones, ER-associated degradation (ERAD) components, and proteins involved in lipid biosynthesis to relieve ER stress ( 47 ). The endoribonuclease activity of IRE1 can also degrade other mRNA sequences through regulated IRE1-dependent decay to reduce protein synthesis in the ER lumen ( 48 , 49 ). While the activation of IRE1 is associated with cell survival mechanisms to relieve ER stress, persistent IRE1 activity can induce apoptosis by activating tumor necrosis factor receptor-associated factor 2, apoptosis signal-regulating kinase 1, and mitogen-activated protein kinases ( 26 , 50 ). Section title: IRE1 is required for placental development in mice Educational score: 4.630021572113037 Domain: biomedical Document type: Study Language: en IRE1α (herein referred to as IRE1) is essential for placental development in mice. Embryos and placentas with a deletion in Ern1 , the gene that encodes IRE1, are smaller than wild-type and heterozygous siblings, and die around embryonic day 12.5 ( 51 – 53 ). Ern1 -/- placentas have reduced blood spaces in the labyrinth zone compared to wild-type mice, accompanied by decreased expression of vascular endothelial growth factor, suggesting that IRE1 contributes to placental vascular development ( 52 – 54 ). There does not appear to be an effect on decidualization in these mice ( 52 ). Interestingly, activation of IRE1 signaling, based on detecting IRE1 phosphorylation and Xbp1 splicing, occurs in the placenta throughout early and midgestation but not in the embryo ( 52 ), suggesting that IRE1 signaling is particularly important for placental development. In line with this observation, midgestation lethality is avoided in mice lacking Ern1 in embryonic tissue but not trophoblasts (using a Mox2 +/Cre transgenic mouse), demonstrating that the cause of embryonic demise in Ern1 -/- mice is due to defective placental development ( 52 ). Therefore, IRE1 appears to have a critical role for placental development in mice. Section title: IRE1 is required for placental development in mice Educational score: 4.320638656616211 Domain: biomedical Document type: Study Language: en In line with an essential role of IRE1 signaling for placentation, high levels of XBP1s are detectable in the mouse placenta, but not in other embryonic organs ( 52 ). XBP1s is also spatiotemporally expressed in the mouse uterus where it is mainly detected in epithelial cells during early pregnancy and then subsequently in DSCs ( 55 , 56 ). To investigate whether canonical IRE1-XBP1s signaling is involved in placental development, Xbp1 -/- mice have been generated. Interestingly, embryos and placentas with a deletion in Xbp1 exhibit embryonic lethality between embryonic day 12.5 and 14.5; however, unlike Ern1 -/- mice, the cause of lethality is attributed to liver hypoplasia. There does not seem to be a notable difference in placental morphology or vascular endothelial growth factor levels between Xbp1 -/- mice and wild-type mice ( 52 , 57 ). Therefore, the essential role of IRE1 in mouse placental development may be through a noncanonical signaling pathway independent of XBP1s. Section title: IRE1 in human STB formation Educational score: 4.474608421325684 Domain: biomedical Document type: Study Language: en There is strong evidence that IRE1 signaling is active and required during human STB formation. Activation of IRE1 signaling may be downstream of cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling, a well-established inducer of trophoblast syncytialization ( 58 , 59 ). PKA phosphorylates IRE1 on Ser 724 ( 58 , 60 ), and also activates cAMP-response element binding protein, which is a transcriptional activator of ERN1 ( 61 ). STB formation in vitro using primary CTBs from term placenta is associated with IRE1 activation. Similar results are observed using BeWo cells, a CTB-like choriocarcinoma line that fuses to form STB-like cells after exposure to forskolin ( 62 ). Interestingly, the addition of small molecule inhibitors targeting IRE1, or RNA-interference targeting all three UPR branches, reduces CTB fusion, hCG secretion, and autophagy ( 62 ). Therefore, similar to its important role in mouse placentation, IRE1 appears to be required for human STB formation. Section title: IRE1 in human STB formation Educational score: 4.433346271514893 Domain: biomedical Document type: Study Language: en Why is IRE1 signaling needed for STB formation? While this answer remains unclear, it may be related to the increased synthesis and secretion of numerous proteins involved in the differentiation of CTBs and their fusion into STBs. Increased demand for protein synthesis during STB formation can exceed the ER’s folding capacity, resulting in the activation of the UPR to adapt to the new protein burden. Since IRE1-XBP1s signaling promotes the production of chaperones and ERAD components, its activation during STB formation may promote protein folding and clearance to ensure proper proteostasis in the ER, thereby enabling STB function. IRE1 activation may also promote the expression of genes involved in phospholipid biogenesis to remodel the ER membrane and expand the ER lumen during STB formation ( 63 , 64 ). Section title: IRE1 in human STB formation Educational score: 4.496169090270996 Domain: biomedical Document type: Study Language: en Apart from responding to ER stress signals, XBP1s transcriptionally regulates the expression of differentiation-associated genes in other cells ( 65 , 66 ). It is therefore possible that XBP1s may exert transcriptional control of genes needed for STB formation and function, such as ER-resident proteins needed for protein processing of STB-related hormones. As one example, the prototypical STB hormone, hCG, is a glycoprotein containing 11 disulfide bonds. These disulfide bonds are essential for hCG to fold into its proper protein configuration ( 67 – 69 ). XBP1s transcriptionally regulates the expression of PDIs, which are needed in the ER for disulfide bond formation and stability of this protein ( 70 ). Furthermore, XBP1s also transcriptionally regulates secretory pathway components, including those involved in protein glycosylation and vesicle trafficking needed for maturation and secretion of hCG. Thus, IRE1-XBP1s signaling may be required for the synthesis, processing, and secretion of hCG, along with other STB-associated proteins ( 71 ). Section title: IRE1 in human STB formation Educational score: 4.36777925491333 Domain: biomedical Document type: Study Language: en CTB fusion involves dynamic changes in membrane composition and membrane-membrane interactions facilitated by syncytins, resulting in the formation of fusion pores, merging of membranes, and intermixing of cytoplasm between adjacent cells ( 72 ). While there is no evidence to-date that IRE1-XBP1s signaling transcriptionally regulates the expression of syncytins or their receptors, a role for IRE1-XBP1s signaling has been identified in other models of cell fusion. For example, deletion of IRE1 in mouse muscle satellite cells inhibits myoblast fusion and impairs skeletal muscle regeneration. IRE1-XBP1s signaling directly controls the expression of several genes involved in myoblast fusion, including Mymk (encoding Myomaker) ( 73 ). IRE1-XBP1s may similarly regulate the expression of genes involved in STB formation; or at minimum, may be needed to produce chaperones and other proteins that facilitate the synthesis, transport, and membrane presentation of fusogens. Section title: IRE1 in human STB formation Educational score: 4.5750203132629395 Domain: biomedical Document type: Study Language: en Aside from direct transcriptional control of fusogens, IRE1 may play a role in cell fusion during STB formation by interacting with filamin A, an actin binding protein that regulates cytoskeletal organization. IRE1 acts as a scaffold by recruiting kinases, such as protein kinase C alpha, to promote phosphorylation of filamin A and induce cytoskeletal remodeling, an essential process that occurs during cell fusion ( 74 – 76 ). Furthermore, IRE1 may facilitate cell fusion by initiating contact sites between the ER and plasma membrane through store-operated calcium channels, which regulate intracellular calcium levels and maintain lipid homeostasis. IRE1 interacts with stromal interacting molecule 1 (STIM1), a sensor of calcium levels in the ER lumen ( 77 ). When calcium levels in the ER lumen are low, STIM1 undergoes a conformational change to promote calcium entry from the cytosol to the ER lumen ( 77 , 78 ). Therefore, by interacting with STIM1, IRE1 may regulate cytosolic calcium levels, which along with increased cAMP, is a requirement for STB formation ( 79 ). Section title: IRE1 in human EVT formation and function Educational score: 4.219586372375488 Domain: biomedical Document type: Study Language: en IRE1 signaling is enriched in first trimester EVTs compared to CTBs, and gene set enrichment analysis of EVTs derived from human trophoblast stem cells shows enrichment of XBP1s-induced chaperones ( 80 ). Furthermore, the same study showed that inhibition of IRE1 using a small molecule inhibitor, 4µ8c, reduces cell surface HLA-G expression, suggesting that IRE1-XBP1s signaling is active during EVT formation and may be required for the synthesis and/or transport of cell surface antigens. Section title: IRE1 in human EVT formation and function Educational score: 4.4585490226745605 Domain: biomedical Document type: Study Language: en While the specific role of IRE1-mediated signaling in EVT formation and invasiveness is not fully understood, interactions with other factors deemed important for EVT function have been noted. For instance, Krüppel-like factor 6 (KLF6) is a transcription factor highly expressed in the placenta that modulates EVT formation and invasiveness. Using HTR-8/SVneo cells, an immortalized cell-line derived from first trimester placental outgrowths and often used as a model of invasive EVTs, silencing KLF6 reduces IRE1 and XBP1s levels, suggesting that KLF6 may be upstream of IRE1 signaling. Interestingly, both KLF6 and IRE1 deficient mouse models are embryonic lethal at embryonic day 12.5, suggesting that they may be part of a similar developmental pathway ( 52 , 81 ). IRE1 may also promote the expression of high-temperature requirement A serine peptidase 1 (HTRA1) following exposure to ER stress conditions, which promotes invasion of HTR-8/SVneo cells ( 82 , 83 ). Notably, in other cells, IRE1-XBP1s signaling promotes the transcription of genes encoding factors associated with epithelial-to-mesenchymal transition, such as SNAI1 , SNAI2 , ZEB2 and TCF3 ( 84 ). EVTs undergo epithelial-to-mesenchymal transition as they gain invasive properties ( 85 ), but further studies are needed to determine whether IRE1-XBP1s signaling may contribute to this aspect of EVT development. Section title: IRE1 signaling during human decidualization Educational score: 4.600564956665039 Domain: biomedical Document type: Study Language: en There is evidence that IRE1-XBP1 signaling is active during decidualization. For example, cytoplasmic and nuclear reactivity for unspliced and spliced variants of XBP1 are detected in both DSCs and glandular epithelium ( 86 ). Decidualization of ESCs is associated with increased levels of IRE1 and its downstream targets, XBP1s and CCAAT/enhancer-binding protein homologous protein (CHOP). Treatment of DSCs with the IRE1 inhibitor, STF-083010, downregulates the expression of CHOP and prevents the secretion of interleukin-1β (IL-1β) associated with decidualization ( 87 ). Furthermore, in a co-culture model designed to mimic implantation, inhibition of IRE1 reduced invasion of trophoblast spheroids (derived from Swan-71 immortalized trophoblasts) into DSCs, suggesting that IRE1 may be required for this process ( 87 ). While IRE1-mediated signaling may be required for decidualization, hyperactivation may induce autophagy and disrupt decidualization. For example, the IRE1-XBP1s pathway is negatively regulated by the heat shock cognate 71 kDa (Hsc70). Knockdown of Hsc70 increases XBP1 protein expression, triggers autophagy, and impairs decidualization of ESCs ( 88 ). These results suggest that moderate activation of IRE1 supports successful decidualization, whereas too little or too much activation can interfere with this process. Section title: IRE1 signaling in the placenta during pregnancy complications Educational score: 4.275423049926758 Domain: biomedical Document type: Study Language: en Aberrant ER stress and hyperactivation of IRE1 signaling is associated with dysregulated placental development and adverse pregnancy outcomes. This has been most frequently documented in pregnancies complicated by preeclampsia and FGR. Compared to placentas from normotensive pregnancies, placentas from preeclamptic pregnancies (particularly early-onset preeclampsia) exhibit increased IRE1 phosphorylation, XBP1 splicing, and ER luminal dilation ( 89 , 90 ). Likewise, expression of XBP1 is increased in decidual tissues from preeclampsia with and without FGR ( 86 ). Endometrial biopsies from patients with recurrent pregnancy loss and implantation failure have elevated expression of IRE1 compared to controls, but surprisingly lower expression of XBP1s, suggesting that dysregulated IRE1 activation may contribute to these pregnancy complications ( 87 ). However, phosphorylated IRE1 was not measured in the samples from recurrent pregnancy loss and implantation failure, and should be evaluated in future studies to determine whether IRE1 activity may be altered in these pregnancy complications. Section title: IRE1 signaling in the placenta during pregnancy complications Educational score: 4.3283891677856445 Domain: biomedical Document type: Study Language: en IRE1 phosphorylation, along with other markers of ER stress and apoptosis, is also increased when subjecting trophoblasts to hypoxia-reoxygenation in vitro to mimic the conditions that placental cells would experience in early-onset preeclampsia ( 91 – 93 ). Therefore, IRE1 signaling likely contributes to the cellular stress response during pathological conditions. Other pregnancy complications associated with ER stress and IRE1 activation include gestational cholestasis, a condition in which bile acids build up in the liver and enter the bloodstream. In a mouse model of gestational cholestasis, increased IRE1 activation is apparent in placental tissue, and this was associated with apoptosis and FGR. Inhibition of IRE1 signaling using 4µ8c prevents trophoblast apoptosis and rescues FGR in mice. Likewise, inhibiting IRE1 signaling prevents cell death in the HTR-8/SVneo cell-line following exposure to deoxycholic acid ( 94 ). Section title: IRE1 signaling in the placenta during pregnancy complications Educational score: 4.247756004333496 Domain: biomedical Document type: Study Language: en Physiological and environmental stressors that increase the risk of pregnancy complications can induce ER stress and activate IRE1 signaling. For example, maternal obesity is a risk factor for various pregnancy complications. Compared to placentas from normal weight pregnancies, obese pregnancies exhibit increased levels of phosphorylated IRE1 and XBP1s in placental tissue ( 95 , 96 ). Palmitate is the most common saturated fatty acid in the body and is detectable at higher levels in the circulation of obese persons. Increased levels of palmitate alter ER morphology, impair invasiveness, and induce ER stress and apoptotic signaling in various human trophoblast cell-lines ( 97 , 98 ). Other environmental stressors associated with ER stress and IRE1 hyperactivation in trophoblasts include viral infections (e.g., ZIKV) and exposure to toxins such as nicotine and ethanol ( 99 – 102 ). In the latter case, only total IRE1 protein levels were assessed, and therefore the contribution of active IRE1-XBP1s signaling to placental function following exposure to toxins remains elusive. Section title: PERK Educational score: 4.693953514099121 Domain: biomedical Document type: Study Language: en PERK detects and responds to ER stress by reducing the amount of protein in the ER lumen through the attenuation of translation ( 103 ). When there is an overaccumulation of misfolded proteins in the ER lumen, PERK homodimerizes and trans-autophosphorylates at Thr 980 , resulting in the activation of its kinase function ( 104 ). PERK will subsequently phosphorylate eukaryotic initiation factor 2α (eIF2α), preventing the assembly of ribosomes at the initiator codon of mRNA transcripts and blocking protein synthesis ( 105 ). As part of the PERK signaling branch, phosphorylated eIF2α promotes mRNA translation of the activating transcription factor 4 (ATF4), which can modulate transcription depending on its binding partners. The classical transcriptional target of ATF4 during ER stress is DDIT3 , which encodes for CHOP, a transcription factor that regulates apoptosis ( 106 ). ATF4 can also transcriptionally regulate genes involved in amino acid metabolism and autophagy ( 107 ). Section title: PERK regulates proper protein folding in the mouse placenta Educational score: 4.296706676483154 Domain: biomedical Document type: Study Language: en Mice lacking PERK are viable and do not initially differ in weight compared to their wild-type counterparts, suggesting that PERK activity is not required (or is redundant) for placental and embryo development ( 108 ). However, other organs with secretory activity are severely impacted by PERK deficiency. For example, mice lacking PERK exhibit progressive loss of pancreatic β cells, neonatal development of diabetes mellitus, and exocrine pancreatic atrophy. Pancreatic cells in these mice have ER lumen dilation and accumulation of electron-dense material in the ER. Mice lacking PERK also have severe skeletal dysplasias and dwarfism associated with reduced hepatic secretion of insulin-like growth factor 1 ( 108 – 110 ). Since the placenta is also a secretory organ, PERK deficiency may affect aspects of placental endocrine function. Section title: PERK regulates proper protein folding in the mouse placenta Educational score: 4.414913177490234 Domain: biomedical Document type: Study Language: en To determine a potential contribution of PERK signaling to placental endocrine activity, a conditional mouse model with PERK depletion specifically in the junctional zone has been investigated. The junctional zone secretes numerous hormones and growth factors, and is therefore considered as the endocrine component of the murine placenta ( 111 ). There are no differences in litter size, placental weight or fetal weight between wild-type mice and those with junctional zone-specific PERK depletion (as expected, since the global PERK knockout also does not show these differences). However, PERK deficiency exacerbates ER stress in the junctional zone when mice are housed in a reduced oxygen atmosphere, which was done to induce tissue hypoxia and ER stress. When exposed to hypoxia, PERK-deficient junctional zone trophoblasts exhibit dilation of the ER cisternae and accumulation of protein aggregates, indicating possible loss of ER homeostasis through aggregation of misglycosylated secretory proteins in the ER ( 111 , 112 ). Furthermore, these trophoblasts have a reduced capacity to stimulate maternal physiological adaptions, such as the induction of glycogenolysis in the liver. Therefore, in the placenta, PERK may promote proper protein folding and processing in the ER, whereas PERK-deficient mice may be more susceptible to proteinopathies under ER stress-inducing conditions. Section title: PERK regulates proper protein folding in the mouse placenta Educational score: 4.3257670402526855 Domain: biomedical Document type: Study Language: en Activation of PERK attenuates translation through the phosphorylation of eIF2α on Ser 51 ( 113 ). A mouse model has been generated in which this serine is mutated to alanine, preventing the ability of eIF2α to reduce translation ( 114 ). These mice die shortly after birth due to hypoglycemia, resulting from a deficiency in gluconeogenesis and loss of pancreatic β cells. In the placenta, eIF2α mutant mice exhibit increased basal translation, correlating with reduced placental and fetal growth compared to mice possessing at least one wild-type copy ( 112 ). Additionally, these placentas have an accumulation of glycoproteins in the junctional zone and reduced labyrinth zone volume, suggesting that eIF2α dysfunction may disrupt placental endocrine function ( 112 ). Section title: PERK signaling in human placental and decidual development Educational score: 4.545761585235596 Domain: biomedical Document type: Study Language: en As a key regulator of protein translation, PERK signaling may contribute to the control of trophoblast differentiation. For example, PERK signaling is activated in BeWo cells during forskolin-induced differentiation as shown through increased levels of phosphorylated eIF2α, ATF4, and CHOP. Exposing BeWo cells to a PERK inhibitor, GSK2656157, inhibits cell fusion, but interestingly there is no effect on secreted hCG levels. Similar results are obtained using primary CTBs from term placentas ( 62 ). Like IRE1, PERK may promote trophoblast fusion through its interaction with filamin A to regulate cytoskeletal remodeling and ER calcium levels ( 115 ). In HTR-8/SVneo cells and JEG-3 cells, another transformed trophoblast cell-line, induction of ER stress using tunicamycin, thapsigargin, or pro-inflammatory cytokines reduces matrix metalloproteinase 2 (MMP2) levels and cell invasion. However, inhibition of PERK restores MMP2 levels ( 116 ). Given the importance of PERK signaling for ER homeostasis in other secretory cells, PERK may maintain cellular integrity in the placenta, particularly under ER stress-inducing conditions. Section title: PERK signaling in human placental and decidual development Educational score: 4.351856231689453 Domain: biomedical Document type: Study Language: en Within the decidua, DSCs from pregnancies deemed healthy express high levels of phosphorylated PERK. Treatment of ESCs and glandular cells with progesterone activates the PERK pathway, as shown by the increased levels of phosphorylated eIF2α, ATF4, and CHOP ( 87 , 90 , 117 ). Progesterone-driven CHOP expression induces several apoptosis markers (e.g., BAX and cleaved caspase-3) in ESCs and glandular cells. Furthermore, BiP/GRP78, CHOP, and cleaved caspase-3 are upregulated during the secretory phase in stromal and glandular regions of healthy human endometrium, confirming that UPR activation and CHOP-mediated apoptosis increase in response to higher progesterone levels ( 117 ). Altogether, these findings indicate that progesterone-driven activation of the PERK/eIF2α/ATF4 pathway, and subsequent CHOP-mediated apoptosis, may regulate endometrial remodeling during the late secretory phase. Section title: PERK signaling in the placenta during pregnancy complications Educational score: 4.294344902038574 Domain: biomedical Document type: Study Language: en PERK signaling may have important roles in determining cell function and cell fate during placental pathologies. For example, in placentas from preeclamptic pregnancies, increased levels of phosphorylated PERK, phosphorylated eIF2α, ATF4 and CHOP are evident, and these proteins appear to localize predominantly to the STB layer ( 118 , 119 ). Increased levels of phosphorylated PERK are also evident in the decidua ( 90 ). Decidual tissue from preeclamptic pregnancies with FGR have increased phosphorylation of eIF2α and ATF4 levels compared to control pregnancies ( 86 , 120 ). These findings suggest that dysregulation of PERK signaling in the decidua may impair decidualization and contribute to the development of pregnancy complications. Section title: PERK signaling in the placenta during pregnancy complications Educational score: 4.322303771972656 Domain: biomedical Document type: Study Language: en Maternal serum from preeclamptic pregnancies activates the PERK pathway in placental explants and HTR-8/SVneo cells, as shown through increased eIF2α phosphorylation and CHOP levels. Cell death was also apparent after explants and cells were exposed to serum from preeclamptic pregnancies, but the specific contribution of PERK signaling to cytotoxicity was not assessed ( 34 ). It is possible that the PERK-mediated cell death observed in placentas from preeclamptic pregnancies occurs as a result of histone deacetylase (HDAC) deficiency ( 121 ). HDACs are essential for trophoblast differentiation and are often downregulated in placentas from preeclamptic pregnancies ( 121 – 124 ). HDAC2 silencing in HTR-8/SVneo cells increases pyroptosis, which was prevented when PERK was knocked down ( 121 ). Therefore, PERK activation may underlie the increased incidence of cell death in placental pathologies. Section title: PERK signaling in the placenta during pregnancy complications Educational score: 4.2969889640808105 Domain: biomedical Document type: Study Language: en To recapitulate the ER stress-inducing conditions that may induce PERK signaling and affect trophoblast function and viability, several in vitro models of cell stress have been devised. For example, exposure of BeWo cells to hypoxia/reoxygenation (fluctuating between room air and 1% O 2 ) leads to increased levels of phosphorylated eIF2α levels and reduced cell number ( 39 ). IL-1β, a pro-inflammatory cytokine that is increased in preeclampsia, induces apoptosis in BeWo cells through the PERK pathway, which is inhibited by progesterone ( 118 ). PERK inhibition can also reduce apoptosis in BeWo cells treated with endocannabinoid 2-arachidonoylglycerol ( 125 ). Cadmium, an environmental pollutant and carcinogen, reduces 11β-HSD2 expression in JEG-3 cells, which is restored either by knocking down PERK expression or by treating cells with antioxidants such as melatonin or N-acetylcysteine ( 126 , 127 ). Thus, signaling through PERK may regulate trophoblast survival under various ER stress-inducing conditions. Section title: ATF6 Educational score: 4.449471473693848 Domain: biomedical Document type: Study Language: en ATF6 is the third ER transmembrane sensor that is activated in response to ER stress ( 128 ). There are two isoforms of ATF6: ATF6α and ATF6β. Signaling through ATF6α generally results in a more potent but transient transcriptional response, whereas ATF6β is a comparatively weaker transcriptional modulator ( 129 ). During ER stress, the cytosolic domain of ATF6 translocates to the Golgi apparatus to be cleaved by site 1 and site 2 proteases ( 130 ). These proteases remove the luminal and transmembrane portions of ATF6, yielding the ATF6 transcription factor. ATF6 binds to the ER stress response element motif to transcriptionally regulate chaperones and ERAD components that enhance protein folding and contribute to clearance of misfolded proteins, respectively ( 131 , 132 ). Section title: ATF6 in mouse placental and decidual development Educational score: 4.48967981338501 Domain: biomedical Document type: Study Language: en While various aspects of the UPR have been studied in the context of placental development, the role of ATF6 remains largely unexplored, highlighting a gap in understanding its potential contributions to trophoblast function and placental health. Knockout of either ATF6α or ATF6β in mice produces viable progeny, but double ATF6α/β knockout mice die early during development (around the time of implantation), suggesting that ATF6α and ATF6β may share an overlapping function that is essential for decidualization and early embryonic development in mice ( 133 , 134 ). In the mouse uterus, expression of ATF6α is primarily localized to luminal and glandular epithelial cells, specifically near the blastocyst implantation site, suggesting a potential role in uterine receptivity ( 135 ). Since ATF6α is also present in primary and secondary decidualization zones, it is plausible that ATF6α could contribute to the progression of decidualization. Indeed, artificial decidualization of pseudopregnant mice increases ATF6α expression in decidual cells ( 135 ). Altogether, increased ATF6α levels in the uterus seem to correlate with the implantation window and initial stages of decidualization in mice, but whether ATF6α directly contributes to these processes requires further exploration. Section title: ATF6 signaling in human placental and decidual development Educational score: 4.358131408691406 Domain: biomedical Document type: Study Language: en ATF6α is present in the human placenta, and staining is particularly strong in nuclei clustered in syncytial knots ( 90 ). Exposure of primary CTBs to an ATF6α inhibitor, AEBSF, reduces cell fusion and hCG secretion ( 62 , 136 ). It is possible that ATF6α promotes STB formation by regulating expression of BiP/GRP78 ( 137 ). Additionally, since ATF6α transcriptionally regulates XBP1 , there may be contributory or compensatory mechanisms through which ATF6α influences STB formation by supporting the IRE1-XBP1s axis ( 131 ). ATF6α is also detected in EVTs, suggesting its participation in the invasion of EVTs into the decidua and interactions with decidual cells ( 86 ). In cancer cells, ATF6α promotes cell invasion and metastasis ( 138 ). Therefore, it is possible that ATF6α may regulate genes involved in EVT migration and invasion, but this requires further investigation. Section title: ATF6 signaling in human placental and decidual development Educational score: 4.202854633331299 Domain: biomedical Document type: Study Language: en Decidual cells from healthy pregnancies exhibit high levels of ATF6α localized primarily to the cytoplasm, indicating the presence of primarily inactive forms of ATF6α ( 90 ). Decidualization of ESCs is associated with an upregulation of ATF6α. Inhibition of ATF6 using AEBSF reduces the decidualization-associated increase in IL-1β secretion, which was also reported when using an IRE1 inhibitor ( 87 , 139 ). It is unclear whether the mechanistic control of inflammatory cytokine production by ATF6α overlaps with the IRE1 signaling pathway. Section title: ATF6 signaling in the placenta during pregnancy complications Educational score: 4.317715644836426 Domain: biomedical Document type: Study Language: en In early-onset preeclampsia with FGR, ATF6α protein levels are increased in placental tissue compared to placentas from pregnancies deemed healthy ( 39 , 86 ). It is unclear if ATF6α activation is also increased, as the levels of active ATF6α, or nuclear localization of the protein, were not assessed. A hallmark of placentas from preeclampsia is reduced secretion of placental growth factor, which is a pro-angiogenic molecule important for maintaining endothelial integrity. Interestingly, reduced placental growth factor in preeclampsia is associated with nuclear localization of ATF4 and ATF6β (but not ATF6⍺ or XBP1) in the STB layer ( 140 , 141 ). Combined ATF4 and ATF6β silencing in BeWo cells exposed to the ER stressor thapsigargin or hypoxia/reoxygenation increased expression of placental growth factor ( 141 ), suggesting that ATF4 and ATF6β negatively regulate placental growth factor expression. Therefore, interfering with ATF4 and ATF6β may be a feasible means to enhance placental growth factor production in compromised pregnancies. Section title: Future applications: targeting ER stress to alleviate placental dysfunction Educational score: 4.542523384094238 Domain: biomedical Document type: Review Language: en As highlighted in the preceding sections, placental development and function require a fine balance of ER stress and UPR activation to maintain cellular homeostasis. Too much or too little UPR activation can impair placental and decidual function and lead to adverse pregnancy outcomes. Therefore, therapeutic approaches targeting ER stress pathways may be promising avenues to restore proper placental function and improve fetal and maternal outcomes ( 34 , 142 ). As one example, tauroursodeoxycholic acid (TUDCA), a bile acid derivative that is currently being evaluated for the treatment for neurodegeneration, alleviates ER stress by acting as a chemical chaperone and reducing the expression of ER stress-related proteins ( 143 ). In a rat model of advanced maternal age, placental insufficiency accompanied by increased ER stress markers (p-eIF2α and CHOP) was observed in aged, pregnant rats. TUDCA treatment administered via drinking water throughout pregnancy reduced ER stress in the placenta and improved placental blood flow and fetal growth ( 38 , 144 – 146 ). TUDCA treatment of pre-implantation embryos can increase the rate of blastocyst formation and enhance the success of implantation and pregnancy in mice ( 147 ). Another example includes phenylbutyric acid, a fatty acid that is naturally produced through fermentation by colonic bacteria, which ameliorates ER stress by acting as a chemical chaperone ( 148 ). Phenylbutyric acid reduces high glucose-induced ER stress in BeWo cells, suggesting that this compound may be useful to reduce placental ER stress in pregnancies with gestational diabetes ( 149 ). Additionally, phenylbutyric acid can also reduce hypoxia-induced apoptosis in HTR-8/SVneo cells by assisting in protein folding and reducing PERK activation ( 150 ). Overall, the use of chemical chaperones to facilitate protein folding and prevent UPR hyperactivation may be a promising avenue to treat placental pathologies and improve fetal outcomes. Section title: Future applications: targeting ER stress to alleviate placental dysfunction Educational score: 4.050795555114746 Domain: biomedical Document type: Study Language: en Future studies could also explore whether selectively targeting one or more UPR pathways is an effective way to improve placental function ( 39 , 151 , 152 ). There are several small molecule inhibitors that specifically target these pathways and are being tested in humans for their efficacy in disease settings like cancer ( 153 – 155 ), but it remains uncertain whether these inhibitors can be safely used during pregnancy. Unintended off-target effects and safe delivery strategies need to be considered. Conversely, since UPR pathways are essential for various aspects of placental and decidual function, it is possible that transiently stimulating one or more pathways may be beneficial to restore ER homeostasis and promote cell survival. More research is required to determine whether ER stress-related pathways can be safely modulated to fine-tune placental ER homeostasis. Section title: Conclusion and perspectives Educational score: 4.043860912322998 Domain: biomedical Document type: Review Language: en In this review, we highlighted the importance of ER homeostasis and UPR signaling for placental and decidual development and function. Although there is resounding evidence of increased ER stress and UPR hyperactivation during pregnancy pathologies, each UPR signaling branch contributes to basic processes needed for placental and decidual function, and these functions may shift depending on cellular conditions . Therefore, tweaking the activity of UPR branches to restore ER homeostasis holds promise as a treatment approach to improve placental function in compromised pregnancies. Section title: Conclusion and perspectives Educational score: 4.46610164642334 Domain: biomedical Document type: Study Language: en Despite significant advances in understanding the role of UPR pathways in placental and decidual development, several gaps remain. It is apparent that UPR functions extend beyond merely responding to stress, as UPR sensors participate in homeostatic mechanisms to facilitate the high demand for protein synthesis and processing in secretory cells. However, the precise roles of each UPR branch in placental and decidual functions are still elusive. Determining their specific contributions can be challenging because there is significant overlap and compensation between the branches and other stress-related pathways. For instance, Ern1- deficient mouse placentas exhibit increased PERK and ATF6 activation, which may mask additional ways that IRE1 regulates placental development and function ( 52 ). As another example, kinases associated with the integrated stress response, which are not connected to the UPR, can activate eIF2α independently of PERK ( 156 ). Therefore, phosphorylation of eIF2α does not necessarily guarantee that the PERK branch of the UPR is active. Additionally, the source of ER stress and UPR activation during placental and decidual development remains elusive. The high demand for protein and lipid production could be contributing to ER stress and UPR activation during placental and decidual development. Section title: Conclusion and perspectives Educational score: 4.490574836730957 Domain: biomedical Document type: Review Language: en Another challenge with assessing ER stress, particularly in the context of placentation, is the limited tools that are available to confirm UPR branch activation. Many studies rely on detecting a handful of changes in gene expression or protein levels of UPR markers in bulk tissue or cell-lines. As the field evolves and better cell models and tools are developed, a more comprehensive understanding of each UPR branch is likely to emerge. For instance, the use of more complex cellular models of the decidua and placenta, such as decidual and trophoblast organoid cultures or “on-a-chip” platforms, offer promising avenues for better representing cellular interactions at the maternal-fetal interface. The use of these models in combination with more precise detection of UPR activation, such as reporter plasmids to measure activation of specific UPR branches in combination with omics technologies for more comprehensive coverage of downstream UPR targets, may provide a deeper understanding of the cellular response to stress at the maternal-fetal interface. The use of CRISPR-mediated gene targeting to disrupt each ER sensor in trophoblast and decidual models will also be enlightening. Collectively, uncovering the role of UPR branches holds potential for a greater understanding of ER homeostasis during normal and pathological placentation. | Review | biomedical | en | 0.999997 |
PMC11695240 | Section title: Introduction Educational score: 3.8937911987304688 Domain: biomedical Document type: Review Language: en Lung cancer is a distinct and heterogeneous disease ( 1 ). Recent decades have witnessed a surge in both morbidity and mortality rates associated with lung cancer. It is ranked first in mortality and second in morbidity among all tumors ( 2 ). The latest global cancer statistics for 2022 indicate that lung cancer remains the foremost cause of death among cancer patients, accounting for 18.7% (1.817million) of all cancer fatalities ( 3 ). The rising number of smokers has precipitated a marked increase in lung cancer incidence ( 4 ). Concurrently, the potential risks of lung cancer in non-smokers (comprising 25% of lung cancer patients) cannot be overlooked ( 5 ). Section title: Introduction Educational score: 3.9309558868408203 Domain: biomedical Document type: Review Language: en Extensive screening and early diagnosis of lung cancer are essential for the prognosis of lung cancer patients. Unfortunately, due to technical bottlenecks, patients with lung cancer often fail to identify the optimal window period for receiving the best treatment, making it a challenging enigma ( 6 , 7 ). Therefore, procedural screening should be provided as early as possible for patients at high risk of lung cancer to prevent invasive development and systemic metastasis. This strategy can help address the complexities of curing advanced types of lung cancer and significantly improve the patient’s prognosis. Currently, there are six available diagnostic methods for detecting lung cancer: chest radiographs (CXRs) ( 8 ), computed tomography (CT) scans ( 9 ), magnetic resonance imaging (MRI) ( 10 ), positron emission tomography (PET) ( 11 ), cytology sputum analysis ( 12 ), and breath analysis ( 13 ). Section title: Introduction Educational score: 3.9421887397766113 Domain: biomedical Document type: Study Language: en Early diagnosis and treatment of lung cancer patients can be significantly advanced through the judicious application of nanotechnology. Nanoparticles (NPs) represent a crucial component of nanotechnology, possessing a unique structure that can amplify the imaging signal of MRI, thereby increasing its detection sensitivity. Moreover, NPs can serve as modification materials for biosensors, enhancing their detection limits and enabling the earlier identification of lung cancer biomarkers. Consequently, this improvement can substantially elevate the rates of early detection ( 14 , 15 ). Section title: Introduction Educational score: 4.014671325683594 Domain: biomedical Document type: Study Language: en Bibliometrics is one of the important methods to objectively measure the impact of academic publications and a discipline dedicated to the statistical and quantitative analysis of published literature, aimed at identifying prevailing issues and scientific trends within a research field ( 16 ). And it can elucidate the interconnections among publications and the relationships between authors and countries by sorting and analyzing relevant data such as keywords, references, authorship, and geographical affiliations of the articles ( 17 ). The body of research concerning nanotechnology and lung cancer is steadily expanding. However, there exists a paucity of comprehensive and detailed quantitative analyses regarding its current state and developmental trajectories. This study aspires to thoroughly and systematically examine the literature on nanotechnology and lung cancer diagnosis over the past nearly two decades. Additionally, it seeks to forecast future research hotspots, directions, and development trends, thereby providing guidance for researchers in project design and experimental research within this domain. Section title: Data sources and search strategies Educational score: 3.5323126316070557 Domain: biomedical Document type: Study Language: en The precision in document type classification within the Web of Science Core Collection (WoSCC) database surpasses that of alternative databases, thereby establishing it as the preferred option for conducting literature analyses. Consequently, this database was selected for our search. The retrieval and data collection of relevant articles pertaining to the application of nanoparticle in lung cancer were handled and completed on December 14, 2023. The search strategy was revealed as follows: [TS= (lung cancer*) OR TS=(lung tumor*) OR TS=(lung neoplasm*)], [TS=(diagnosis*) OR TS=(Diagnosis and Examinations)], TS=nanoparticle*. Subsequently, we concatenated the above three using the Boolean logic operator “AND” and set the year limit at retrieval to January 1st, 2006, to December 14st, 2023. The document types were Articles and Review Articles. What’s more, the publications were not only unretracted but also not in the “Expression of Concern”. Section title: Inclusion and exclusion criteria Educational score: 3.818436622619629 Domain: biomedical Document type: Study Language: en This study included literature on nanotechnology and lung cancer diagnosis published in different English-language academic journals, including articles and review articles. The exclusion criteria were: meeting abstracts, conference presentations, letters, repeated publications, and unrelated articles. Two reviewers independently sifted through the literature and data. After screening, a total of 966 usable articles were obtained. The data collection and retrieval strategy were shown in Figure 1 . Section title: Software analysis Educational score: 2.0287652015686035 Domain: biomedical Document type: Study Language: en The analysis and visualization of the annual publication trends and proportions of national papers in this study rely on GraphPad Prism v9.4.0. Additionally, CiteSpace version 6.2.R4(64-bit) (Drexel University, Philadelphia, PA, USA) and VOSviewer version 1.6.20 (Leiden University, Leiden, Netherlands) were utilized for further analysis of the data and for visualizing the scientific knowledge atlas. Section title: Software analysis Educational score: 3.7115635871887207 Domain: biomedical Document type: Study Language: en VOSviewer, a free JAVA-based software created by Waltman L and van Eck NJ in 2009, can be used to analyze large volumes of literature data and visualize it in map format ( 18 ). VOSviewer is a software tool designed for constructing and visualizing bibliometric networks. It has been widely used in the fields of bibliometrics and scientometrics to analyze and visualize relationships among scientific publications, authors, institutions, and keywords. VOSviewer enables users to create visual representations of various types of networks, including citation networks, co-authorship networks, and keyword co-occurrence networks. These visualizations help to illustrate the connections and relationships between different scientific entities. By analyzing bibliometric data, VOSviewer can help identify research trends, emerging fields, and influential publications or authors. The software can identify clusters of related publications, authors, or keywords, providing insights into how particular themes or areas of study are connected within the broader context of scientific research. In this study, we applied VOSviewer 1.6.20 to conduct network diagram and density map of the number of article publishment in journals and the keywords. Section title: Software analysis Educational score: 3.8672494888305664 Domain: biomedical Document type: Study Language: en CiteSpace, developed based on Java, is a bibliometrics software to visualize research achievements in a particular field through drawing co-citation network maps ( 19 ). The software aims to use an experimental framework to study new concepts and evaluate existing technologies, enabling users to gain a better understanding of knowledge domains, research frontiers, trends, and predict future research advancements. CiteSpace allows users to perform comprehensive bibliometric analyses, helping to evaluate citation patterns, authorship trends, and the influence of specific publications within a given field. CiteSpace can create co-citation networks that show how often two or more documents are cited together, as well as co-authorship networks that display collaborations between researchers. This feature aids in understanding collaborative research dynamics. In addition, the tool provides temporal visualizations that highlight trends and changes over time within a specific research area. CiteSpace is equipped to identify emerging research frontiers by analyzing keywords and their occurrences in the literature. In this study, we utilized CiteSpace 6.2.R4(64-bit) to conduct cluster analysis and collaborative network analysis of authors, institutions, journals, literature and countries/regions. Section title: Results Educational score: 3.2509260177612305 Domain: biomedical Document type: Study Language: en The results show that from January 1, 2006, to December 14, 2023, a total of 966 articles on the application of nanoparticles in lung cancer were found in the WoSCC database. This includes 711 (73.6%) articles and 255 (26.4%) reviews. The literature covers 73 countries and regions, 1552 institutions, and 5240 authors. Section title: Analysis of annual published papers Educational score: 3.685520887374878 Domain: biomedical Document type: Review Language: en Since 2006, there has been a gradual increase in the annual publication count within the domain, as illustrated in Figure 2 . This timeline has been segmented into three distinct phases for analytical clarity. The initial phase, from 2006 to 2013, exhibited a modest growth in publications, with fewer than 30 papers being published annually, reflecting limited engagement from the research community. The subsequent phase, from 2014 to 2019, was characterized by a steady uptick in publication numbers, denoting an escalating interest and visibility of the field within the academic circles. The period following 2020 marked a significant surge in publication volume, culminating in a peak in 2022, which signifies a broad recognition and intensive exploration of the field post-2020. Overall, the utilization of nanoparticles in lung cancer diagnostics is garnering escalating interest and is under constant advancement. Section title: Global publications and cooperation analysis of different countries/regions and institutions Educational score: 3.7650365829467773 Domain: biomedical Document type: Study Language: en As of the specified data collection deadline, investigations into the application of nanoparticle for lung cancer diagnostics have been conducted in 73 distinct countries and regions. Table 1 shows the top 10 countries/regions ranked according to their publication volume, and the top five countries in this field are China, the United States, India, South Korea, and Iran. China contributed the most published papers (452, 46.12%), followed by the USA (172, 17.55%), and India (108, 11.02%). The heat map and line chart plotted by the number of publications issued by each country are shown in Figure 3 . The number of publications of the USA has remained at a dozen, with a peak occurring in 2018 (20), while India’s publications peaked in 2022 (27). In contrast, China’s publications have been surging since 2015, and as of the present, China has already published 79 papers in 2023. Section title: Global publications and cooperation analysis of different countries/regions and institutions Educational score: 1.2150813341140747 Domain: other Document type: Other Language: en Among the top 10 countries/regions by publication volume, China and the United States stand out significantly, with their papers receiving 12,827 and 12,405 citations respectively, surpassing the other nations by a wide margin. China leads in both publication volume and the number of citations, yet its citation per publication ratio is 28.38, ranking only seventh. Conversely, the United States, securing the second position in terms of both publication volume and citation count, boasts a citation per publication ratio of 72.12, which is the second-highest, reflecting a superior overall quality of their research outputs. Section title: Global publications and cooperation analysis of different countries/regions and institutions Educational score: 2.500988721847534 Domain: biomedical Document type: Study Language: en The institutions ranked in top 10 for publication volume are as shown in Table 2 . A total of 1552 institutions systematically published articles on the application of nanoparticle in diagnosis of lung cancer. In terms of the leading research institutes, The Chinese Academy of Sciences topped the list with 59 publications and 1759 citations, while Shanghai Jiao Tong University and Fudan University (18 publications and 688 citations) were the top three institutions in terms of the highest number of publications in China, holding the top four positions. The Technion Israel Institute of Technology in Israel held the third position with 27 publications, however, its notable citation frequency of 3,601 resulted in an impressive average citation of 133.37, positioning it at the forefront in terms of citation impact. Section title: Global publications and cooperation analysis of different countries/regions and institutions Educational score: 1.6310994625091553 Domain: other Document type: Other Language: en Figure 4 presents the cooperation analysis of different countries/regions and institutions. As shown in the picture, China have a large number of publications and high citation frequency, while its centrality index is 0.35, indicating that it is the leading country in this field at present. China cooperates more closely with India, South Korea, Turkey, and Israel, while the United States cooperates more closely with Iran, France, and Spain. This indicates academic cooperation between countries exhibits regional characteristics. Upon further analysis, we discovered that research institutions, regardless of country, have a propensity for collaborating with institutions situated within their respective countries. As a result, we advocate for an intensification of cooperation among institutions at both the domestic and international levels to eradicate the barriers obstructing academic collaboration. Section title: Publications, cooperation and co-citation analysis of journals and authors Educational score: 2.4896161556243896 Domain: biomedical Document type: Study Language: en Table 3 presents the top 10 journals by publication volume, as illustrated by the density map in Figure 5A . The Biosensors & Bioelectronics (34 articles, 3.52%) is the journal with the greatest quantity of publications in this field, followed by Sensors and Actuators B-Chemical (31 articles, 3.21%), ACS Applied Materials & Interfaces (21 articles, 2.17%), and International Journal of Nanomedicine (21 articles, 2.17%). All top 10 publication journals are classified in JCR-Q1, with biosensors & bioelectronics having the highest IF (12.6). Section title: Publications, cooperation and co-citation analysis of journals and authors Educational score: 2.5026581287384033 Domain: biomedical Document type: Study Language: en The impact of a journal is determined by the frequency that was cited by others, indicating whether the journal has had a significant impact in this field. The top 10 co-citation journals are exhibited in Table 4 , and Figure 5B shows the visualization map of journals co-citation. There were five journals with more than 350 co-citations, and all of them were in JCR-Q1. ACS Nano was the most frequently co-cited journal (488 times), followed by the ACS Applied Materials & Interfaces (419 times), the Journal of the American Chemical Society (399 times), the Biomaterials (396 times), and the Analytical Chemistry (381 times), while the Biomaterials in the 5th place had a greater centrality and the Advanced Materials in the 9th place possessed a higher IF (29.4). It demonstrated that Biomaterials played an important role in the development of this subject area. Section title: Publications, cooperation and co-citation analysis of journals and authors Educational score: 3.1770715713500977 Domain: biomedical Document type: Study Language: en The distribution of topics in academic publications is displayed through dual overlap map . The colored lines represent the connections between citations, with citing domains on the left and cited domains on the right. Based on the displayed results, we identified 4 main colored citation paths, namely that research publications in the fields of physics/materials/chemistry being cited mainly by research publications in the fields of molecular/biology/genetics and chemistry/materials/physics. Research publications in the field of molecular/biology/immunology is mainly cited by research publications in the fields of molecular/biology/genetics and chemistry/materials/physics. It can be seen that the application of nanoparticle in lung cancer diagnosis involves several main realms such as materials, chemistry, biology, molecular, and immunology. Section title: Publications, cooperation and co-citation analysis of journals and authors Educational score: 3.941906690597534 Domain: biomedical Document type: Study Language: en Table 5 represents the top 10 authors with the greatest number of publications and co-citations, respectively. The top 10 authors published a total of 98 papers, accounting for 10.14% of all articles in the field. Haick, Hossam has the largest quantity of publications (24 articles), followed by Cui, Daxiang (11 articles) and Yang, Huaixia (10 articles). Among the top 10 ranked authors, six are from China, two from Israel, and two from South Korea. The network map based on cooperation between authors is shown in Figure 6A . A knowledge map of author co-citation analysis was displayed in Figure 6B . There are a total of 16 authors who have been cited over 50 times, indicating that the results published by these researchers had had a strong impact on the realm that the application of nanoparticle in lung cancer diagnosis. Among them, Jemal, A, with the biggest node, was the most cocited author (111 times), followed by Zhang, Y (98 times), Siegel, RL (95 times), Peng, G (90 times), and Wang, J (79 times). The above data revealed that Jemal, A held a prominent position in the realm of application of nanoparticle in lung cancer diagnosis. Section title: Visualization and cluster analysis of co-cited references Educational score: 4.254655838012695 Domain: biomedical Document type: Study Language: en By setting the selection criteria as g-index(k=25), LRF=3.0, L/N=10, LBY=5, and e=1.0, the co-cited reference co-occurrence visualization network consisting of 995 nodes and 3346 connections in Figure 7A was obtained, in which the size of the nodes represented the frequency, and the thickness of the lines represented the closeness of the connection. In this figure, the color of the nodes represents the year of publication, with larger nodes indicating that the article has been cited more frequently. The greater the number of connecting lines, the higher the recognition of the article within the field. The article “Cancer Statistics, 2021” in the CA: A Cancer Journal for Clinicians (IF=254.7) is the reference with the highest number of co-citations, with Siegel, Rebecca L. as the first author. The American Cancer Society annually estimates the number of new cancer cases and deaths in the United States and compiles the latest data on population-based cancer occurrence. This article expresses that the mortality rate of cancer, especially lung cancer, is decreasing year by year, with the annual decline rate of lung cancer mortality in male having increased from 3.1% to 5.5%, while that in female having increased from 1.8% to 4.4%, and overall mortality rate from 2.4% to 5%. The 2-year relative survival rate of NSCLC has increased from 34% to 42%, while the survival rate of small cell lung cancer remains 14% - 15%. The innovation of diagnostic and treatment methods has accelerated the development of lung cancer treatment, especially the application of nanoparticles, which has opened up a new avenue for the diagnosis and treatment of NSCLC, and making a series of progress, promoting the reduction of the overall mortality rate of cancer.The second article is “Assessment, origin, and implementation of breath volatile cancer markers”, published by Haick,Hossam, which proposes that cancer can be diagnosed by detecting volatile organic compounds (VOCs) in exhaled air samples, introducing a new method that is non-invasive and potentially cost-effective. Breath analysis is a very young field of research and faces challenges. Nanoparticles can be applied to perform targeted analysis of VOCs related to cancer. The visual map of co-cited references cluster analysis presented in Figure 7B is obtained by log-likelihood ratio (LLR) algorithm, and the details were listed in Table 6 . The top 12 clusters included #0 lung cancer, #1 deep tissue imaging, #2 volatile organic compounds, #3 nanoparticle toxicity, #4 nanotheranostics, #5 biosensors, #6 SERS(Surface Enhanced Raman Scattering), #8 NSCLC(Non-small Cell Lung Cancer), #9 electrospinning, #10 paclitaxel, #11 targeting molecules, and #12 genetically engineered mouse model. Figure 7C shows that deep tissue imaging(cluster 1), nanoparticle toxicity(cluster 3), NSCLC(cluster 8), paclitaxel(cluster 10) are early research hotspots, and volatile organic compound(cluster 2), electrospinning(cluster 9), targeting molecules(cluster 11), genetically engineered mouse model(cluster 12) are mid-term hotspots, and nanotheranostics(cluster 4), biosensors(cluster 5), SERS(cluster 6) represent the hot topics and trends in the field. The horizontal axis represents the year, and a higher shape indicates a greater level of prominence for the topic. Section title: Visualization and cluster analysis of key words Educational score: 4.132208824157715 Domain: biomedical Document type: Study Language: en Table 7 listed the top 10 keywords by frequency and link strength, and their visual maps are respectively shown in Figures 8A and 8B . According to the co-occurrence of keywords in VOSviewer, and as the frequency of co-occurrence is indicative of its popularity, “drug-delivery”, “ in-vitro ”, “delivery”, “therapy”, “breast-cancer”, “biomarkers”, “chemotherapy”, “cells”, “expression”, and “iron-oxide nanoparticles” had been identified as top 10 hot keywords. This suggests that current research on nanoparticles in lung cancer predominantly concentrate on their application in drug delivery, with the objective of utilizing this approach for therapeutic interventions. We removed irrelevant keywords and constructed a network of 182 keywords which appeared at least 28 times, resulting in four different clusters. The first cluster is red, with 63 keywords, including in-vitro 、drug-delivery、chemotherapy、nanomedicine、tumor microenvironment、magnetic nanoparticles, targeted delivery, co-delivery, photodynamic therapy, imaging; the second cluster is green, with 52 keywords, including DNA, SERS, biosensor, immunoassay, quantum dots, sensitive detection, spectroscopy, aptamer, fluorescence, amplification, assay, platform, graphene; the third cluster is blue, with 43 keywords, including delivery, therapy, cells, expression, biomarker, cytotoxicity, apoptosis, resistance, metastasis, exosm, activation, migration, oxidative stress; and the fourth cluster is yellow, with 28 keywords, including biomarkers, carbon nanotubes, gas sensors, breath, array, surface, disease, acetone. We utilized CiteSpace to create a volcano plot, thereby visually illustrating the temporal evolution of research hotspots . The figure reveals that “photodynamic therapy”, “biosensors”, and “silver nanoparticles” have remained consistently prominent research themes in this domain. Section title: Strongest citation burst of references and key words Educational score: 1.7264753580093384 Domain: biomedical Document type: Other Language: en Burst analysis is a method that identifies research hotspots and emerging frontiers within a specific period. Figures 9 illustrate the intensity of these bursts, along with their respective start and end times. These visualization diagrams are generated by CiteSpace. Section title: Strongest citation burst of references and key words Educational score: 3.9612019062042236 Domain: biomedical Document type: Study Language: en Figure 9A enumerates the top 50 keywords exhibiting the most pronounced citation bursts. The initial phase of research predominantly centered on “biomarkers” , “gold nanoparticles” , and “magnetic nanoparticles” . This trend underscores an early emphasis on the application of nanomaterials and biomarker detection. In subsequent years, the emergence of terms such as “chitosan nanoparticles,” “targeted drug delivery,” “targeted therapy,” and “tumor microenvironment” illustrates a shift towards a more nuanced focus on the tumor microenvironment and targeted therapeutic delivery. This evolution highlights the burgeoning synergy between nanomaterials and biotechnology as a pivotal area of research. Furthermore, the dynamic progression of research themes over time attests to the ongoing advancements in nanomaterial technologies and innovative strategies for lung cancer diagnosis, indicating a positive developmental trajectory in this domain. Section title: Strongest citation burst of references and key words Educational score: 1.4875186681747437 Domain: biomedical Document type: Other Language: en In terms of the top 50 references exhibiting the most significant citation bursts, Peng G’s 2009 publication in Nature Nanotechnology and a 2010 paper in the British Journal of Cancer , along with Blanco E’s 2015 publication in Nature Biotechnology , and Amreddy N’s 2018 paper in Nanomedicine: Nanotechnology, Biology, and Medicine , have covered a period of time until 2023. The research content in these articles is consistent with the key concerns reflected in the current burst keywords. Section title: Discussion Educational score: 3.808515787124634 Domain: biomedical Document type: Study Language: en Over the past nearly two decades, the number of studies on nanoparticles in the field of lung cancer diagnosis has continuously increased, exhibiting an overall upward trend. From 2006 to 2013, research on nanoparticles in lung cancer diagnosis was in its nascent stages, with an average annual publication fewer than 30 articles. Subsequently, from 2014 to 2023, research in this field has been significantly strengthened, with an average annual publication 89.1 articles, peaking in 2022 with 145 articles. This trend indicates a growing involvement of researchers in the study of nanoparticles for lung cancer diagnosis. Notably, China leads in publication number, contributing 452 articles, accounting for 46.79%. Section title: Discussion Educational score: 3.905167579650879 Domain: biomedical Document type: Study Language: en The number of research papers in this field is concentrated in economically strong countries. The h-index and the number of citations are frequently utilized to evaluate the academic standing of a country or region ( 20 – 22 ). The United States pioneered the application of nanomaterials in lung cancer diagnosis and has yielded prolific research outcomes. Between 2006 and 2010, more than half (52.9%) of the publications were published by them. Over the past decade, starting from 2014, China has experienced a significant surge in publication number, reflecting increased research investment in the application of nanomaterials in lung cancer diagnosis and expanding collaboration with various research institutions both domestically and internationally, thereby propelling the global advancement of this field. Extensive international scientific collaboration contributes to the maturation of the research field ( 23 ). However, the citation-to-publication ratio of Chinese papers ranks only seventh among all countries, suggesting that the quality of research still requires enhancement, indicating a discrepancy between quantity and quality. Section title: Discussion Educational score: 1.0555962324142456 Domain: other Document type: Other Language: en Regarding institutional contributions, the Chinese Academy of Sciences and Shanghai Jiao Tong University rank first and second in publication number, respectively. However, the Technion Israel Institute of Technology, which ranks third, boasts the highest total citation volume and average citation volume, signifying that its research in this field is widely acknowledged and esteemed worldwide. Section title: Discussion Educational score: 4.137883186340332 Domain: biomedical Document type: Study Language: en After excluding irrelevant keywords, we constructed a network comprising 182 keywords, each appearing at least 28 times, thereby delineating four distinct clusters. Notably, “biomarkers” emerged as the sixth most frequent keyword and constitutes a pivotal element of the yellow cluster, intricately associated with other terms, thereby underscoring its indispensable role in early cancer diagnosis. Biomarkers have persistently been a focal point in lung cancer diagnostics. The oncogenic process is frequently accompanied by mutations in DNA and RNA, aberrant protein expression, and methylation or point mutations of organic compounds such as cytokines. Many of these alterations can be detected months or even years before clinical diagnosis, and are thus identified as cancer biomarkers ( 24 – 26 ). Consequently, utilizing cancer biomarkers for preventive lung cancer diagnosis in high-risk cohorts can preclude the physical detriments associated with radiological examinations and pathological biopsies. Moreover, this approach mitigates the secondary trauma inflicted by invasive tests. Section title: Discussion Educational score: 3.9594497680664062 Domain: biomedical Document type: Review Language: en Comprehensive research reveals that biomarkers are predominantly classified into two categories: protein and genetic biomarkers ( 27 ). Various cancer biomarkers have been identified for lung cancer detection, including carcinoembryonic antigen (CEA) ( 28 ), cytokeratin fragment 21-1 (CYFRA21-1) ( 29 ), carbohydrate antigen 125 (CA125) ( 30 ), transthyretin (TTR) ( 31 ), haptoglobin ( 32 ), neuron-specific enolase (NSE) ( 33 ), GM2 activator protein (GM2AP) ( 34 ), carbohydrate antigen 19-9 (CA19-9) ( 35 ), p16 ( 36 ), and the KRAS ( 37 ). Section title: Discussion Educational score: 4.103169918060303 Domain: biomedical Document type: Study Language: en Among these, CYFRA21-1 and NSE are extensively utilized for lung cancer diagnosis. Moreover, these biomarkers serve as differentiators between non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), significantly aiding in the precise diagnosis and classification of lung cancer subtypes ( 38 , 39 ). Previous studies have corroborated the high specificity and sensitivity of CYFRA21-1 as a biomarker in diagnosing NSCLC, with its efficacy in detecting squamous cell carcinoma being particularly notable ( 40 ). Additionally, CYFRA21-1 has demonstrated substantial utility in the therapeutic management of lung cancer patients. Research consistently indicates that CYFRA21-1 levels can predict chemotherapy efficacy in patients with advanced NSCLC. Recent investigations have further substantiated this correlation, revealing that the rate of change in CYFRA21-1 levels before and after the initial chemotherapy cycle inversely correlates with chemotherapy efficacy ( 41 , 42 ). Section title: Discussion Educational score: 3.9370501041412354 Domain: biomedical Document type: Study Language: en Sometimes, a single cancer biomarker may not suffice to achieve a 100% confirmation diagnosis rate for early-stage lung cancer patients; combining multiple lung cancer biomarkers can significantly enhance the diagnostic rate for these patients ( 43 ). There are researches have shown that the sensitivity of using a combination of CEA, CYFRA21-1, and NSE as biomarkers is higher than that of using only two or one biomarker. Moreover, the sensitivity of integrating tumor biomarkers with imaging studies for early lung cancer diagnosis reaches up to 90% in clinical case analyses ( 44 ). However, the statistical analysis only covered 180 patients, and its reliability needs further validation. Section title: Discussion Educational score: 3.949125051498413 Domain: biomedical Document type: Study Language: en In the keyword clustering volcano plot, “biosensors” emerge as critically important. Researchers and clinicians frequently employ immunological techniques for biomarker analysis, such as the conventional enzyme-linked immunosorbent assay (ELISA), to identify substances like antigens and antibodies. This method is renowned for its exceptional sensitivity and specificity, rendering it an excellent detection tool; however, its complexity and cost restrict its broad applicability ( 45 , 46 ). Consequently, the development of a portable and rapid biosensor for detecting lung cancer biomarkers could significantly improve early lung cancer diagnosis rates. Section title: Discussion Educational score: 4.049563407897949 Domain: biomedical Document type: Study Language: en Biosensors consist of two primary components: sensors and biometric elements, which capture and react with a series of biomarkers, subsequently converting biochemical reactions into measurable signals to provide clinical personnel with patient testing information expeditiously ( 38 , 47 ). The performance of biosensors is typically assessed using two metrics: the detection limit and sensitivity. A lower detection limit and higher sensitivity correlate with superior biosensor performance ( 48 ). For instance, a recent study reports that immunosensing of NSE, a standard lung cancer biomarker, employs a nanocomposite of mesoporous silica encapsulated with CuO2 nanoparticles to develop an innovative electrochemiluminescence sensing platform. This method shows promise as a cost-effective approach to detecting neuron-specific enolase antigen in serum ( 49 ). Additionally, considering the specific site of cancer in the lung, detecting volatile compounds in exhaled breath presents a viable approach, and the development of nano-biosensors could also be targeted towards this research area ( 50 ). Section title: Discussion Educational score: 3.687885046005249 Domain: biomedical Document type: Study Language: en Although theoretical research on biosensors is abundant, several challenges persist in their practical design and clinical application. For example, the receptor on the recognition element may rapidly deteriorate, reducing recognition sensitivity. Consequently, the biomarker may not be easily recognized due to alterations when removed from its native environment ( 51 ). Therefore, further efforts are necessary for researchers to develop biosensors that can meet the demands of large-scale clinical lung cancer detection. Section title: Discussion Educational score: 3.492079019546509 Domain: biomedical Document type: Study Language: en Nanoparticles, as a distinctive application of nanomaterials in emerging nanotechnology, have become significant research subjects, particularly those nanoparticles utilizing precious metals as the material source. These materials exhibit unique physicochemical properties and possess a high surface area-to-volume ratio, which enhances the detection performance of conventional biosensors ( 52 , 53 ). Section title: Discussion Educational score: 3.923755168914795 Domain: biomedical Document type: Study Language: en Gold nanoparticles and silver nanoparticles both emerge in the keyword clustering analysis, highlighting their significant roles in lung cancer diagnostics, with “gold nanoparticles” appearing earlier. Many researchers have found that gold nanoparticles are biocompatible due to their chemical inertness. Their high electrical conductivity, density, and surface area-to-volume ratio distinguish them from other materials. Furthermore, the organic combination of gold nanoparticles and biosensors can effectively improve the detection limit and sensitivity of the sensors ( 54 , 55 ). Section title: Discussion Educational score: 4.123435974121094 Domain: biomedical Document type: Study Language: en Pirzada et al. ( 56 ) developed an ultrasensitive electrochemical sensor using AuNPs-modified epitope-mediated hybrid molecularly imprinted polymers (MIP). Experiments showed that AuNPs hybridized MIP improved the sensor’s sensitivity and expanded the detection range to identify NSE in human serum in the concentration range of 25–4000 pg/mL, effectively enhancing the early detection rate of SCLC. Furthermore, Zeng et al. ( 57 ) developed an ultrasensitive electrochemical immunosensor to detect CYFRA21-1 in human serum and enhance the screening of NSCLC. The team used AuNPs/Thi/MWCNT-NH2 nanocomposites to immobilize horseradish peroxidase-labeled anti-CYFRA21-1. The high anti-CYFRA 21-1 loading capacity, complemented by the good biocompatibility and conductivity of the nanomaterials, allowed the biosensor to have a high linear range of 0.1–150 ng·mL^-1 and a low detection limit of 43 pg·mL^-1. Section title: Discussion Educational score: 3.980203151702881 Domain: biomedical Document type: Study Language: en In addition to gold nanoparticles, sliver nanoparticles have attracted researchers’ interest for their unique antibacterial properties, thermal stability, electrical conductivity, and catalytic activity. They have applied AgNPs in biosensors’ fabrication to enhance lung cancer biomarkers’ detection limits ( 58 , 59 ). Lee et al ( 60 ). used a mixture of silver nanoparticles and reduced graphene oxide-modified screen-printed electrodes to prepare a sensing matrix. They also used horseradish peroxidase-labeled antibodies as recognition molecules for CEA, resulting in a sandwich-type electrochemical immunosensor that performs better in detecting CEA in a simple, rapid, and low-cost manner. Magnetic nanoparticles can also be used in lung cancer diagnostics ( 61 ). However, the current research depth and refinement are insufficient to obtain nanomaterial-derived biosensors with excellent performance. Section title: Discussion Educational score: 4.192434310913086 Domain: biomedical Document type: Study Language: en However, nanoparticles may have potential toxic effects on the human body. Studies have reported that Ag nanoparticles can induce intracellular DNA damage through the GADD45a gene ( 62 ). Besides, there is a study shows that the induced DNA damage and activated caspase-3, p53, p38 and ERK expression by Au/Ag NPs offered leads to their higher cytotoxicity and redox modulations (within mitochondrial membranes) ( 63 ). The toxicity of Au is often greater than that of Ag. For example, one particular investigation examined the effects of both Au and Ag NPs on A549 cell line at 24-hour interval through which the concerned scientists noted that 29.4 μg/mL as IC50, inhibitory concentration for Ag NPs whereas for Au NPs, this value was 49.8 μg/mL ( 64 ). The particle size is one of the factors responsible for showing toxicity of Au NPs, specifically the smaller ones. This is due to their ability to cross the cell membrane and reach the nucleus more rapidly ( 65 ). Not only the size, but also the quantity of nanoparticles and whether they are loaded with other biological materials can influence their toxicity ( 66 ). Albumin-modified gold nanoparticles and chitosan functionalized silver nanoparticles have been demonstrated to possess low in vivo toxicity ( 67 , 68 ). Furthermore, how to synthesize these nanomaterials and how to remove organic solvents, reagents, or toxic chemicals from the reaction mixture are significant challenges we face ( 69 ). Section title: Discussion Educational score: 3.2510058879852295 Domain: biomedical Document type: Other Language: en Gold (Au) and silver (Ag) are categorized as inorganic materials, akin to other inorganic nanomaterials such as silica, hydroxyapatite, and similar calcium-based substances. Conversely, organic materials encompass liposomes, polymers, and carbon-containing compounds. These materials are employed for drug loading and improve therapeutic and diagnostic efficacy via targeted delivery mechanisms. Currently, nanoparticle formulations currently approved by the U.S. FDA for clinical trial include (The registration ID in the U.S. clinical trial database): Section title: Discussion Educational score: 1.5321279764175415 Domain: biomedical Document type: Other Language: ca 1. Inorganic substances: Section title: Discussion Educational score: 2.2046852111816406 Domain: biomedical Document type: Other Language: ca 2. Polymeric micelles nanoparticles: Section title: Discussion Educational score: 3.995650291442871 Domain: biomedical Document type: Review Language: en Scientists have extensively investigated targeting and permeation, including magnetic nanoparticles ( 70 ), thermosensitive nanoparticles ( 71 , 72 ), and pH-sensitive nanoparticles ( 73 ). Simultaneously, scientists have employed tumor cell membranes to encapsulate nanomaterials, enhancing drug delivery efficiency. The aforementioned studies predominantly focus on intravenous administration. Additionally, inhalation therapy emerges as a promising approach for the diagnosis and treatment of lung cancer. Compared to conventional intravenous administration, inhalation therapy provides several advantages, including enhanced pulmonary targeting and reduced systemic drug concentrations, thereby minimizing biological toxicity ( 74 ). Section title: Discussion Educational score: 4.142037391662598 Domain: biomedical Document type: Review Language: en The precision targeted therapy for lung cancer based on nanotechnology aims to minimize the toxic effects of drugs, enhance the efficacy of anticancer chemotherapy agents, and improve tumor imaging. In recent years, this research field has significantly expanded, contributing to the improvement of patients’ quality of life and overall survival rates. Nanoparticles, as excellent biomaterials, exhibit diverse properties that make them suitable for drug delivery applications. They provide sufficient space and protection for drug molecules, preserving their integrity during systemic circulation and preventing exposure to non-target tissues. Furthermore, their surfaces can be functionalized with various targeting moieties to selectively target cancer cells and tumors. Inhaled nanoparticle therapy represents a promising treatment strategy. However, inherent cytotoxicity of anticancer drugs raises concerns regarding pulmonary tolerance, as well as the potential risks of local pulmonary toxicity and adverse reactions. In summary, whether through intravenous administration or inhalation, nanoparticles hold great promise in the treatment of lung cancer. Section title: Discussion Educational score: 2.353090524673462 Domain: biomedical Document type: Study Language: en This study is the first to analyze the application of nanoparticles in lung cancer diagnosis using the bibliometrics method, which has certain guidance and pioneering. However, this study has some limitations. Data sources were obtained from the Web of Science Core Collection only. We only analyzed English studies. Section title: Conclusion Educational score: 4.023190498352051 Domain: biomedical Document type: Study Language: en This study represents the pioneering effort in literature visualization analysis concerning the application of nanoparticles in lung cancer diagnosis using CiteSpace. By conducting a thorough examination of Author/Country/Institutions/Cited Journals/Keyword, we have initially identified and introduced a series of research hotspots and emerging frontiers, including “biomarkers”, “biosensors”, “gold nanoparticles”, and “sliver nanoparticles”. Nevertheless, our analysis was confined to English-language literature within the Web of Science database, which introduces certain limitations. Future investigations in this domain should focus on the synergistic use of various nanoparticles and biosensors to enhance lung cancer detection efficacy and sensor longevity, as well as to delineate the strengths and weaknesses of each material-modified sensor. Moreover, the integrated application of biomaterials and nanomaterials is likely to emerge as a significant research frontier. | Review | biomedical | en | 0.999996 |
PMC11695273 | Section title: Introduction Educational score: 4.296082496643066 Domain: biomedical Document type: Review Language: en Natural hybridisation and introgression occur in at least 10% of animal species and can play a significant role in increasing genetic diversity or creating novel genotypes that affect hybrid fitness . Despite its potential for incompatibilities and deleterious fitness consequences, hybridisation may allow individuals to more quickly occupy new ecological niches or embark on novel evolutionary trajectories . Although adaptive introgression appears to be common and most introgressed variation is selected against , preferential introgression of locally adapted alleles into hybrids may facilitate increased fitness . Indeed, when compared to parental populations, hybrids may exhibit a fitness benefit when challenged with novel environmental conditions, for example, during range expansion , invasion , pathogen exposure or anthropogenic impacts . Such challenges for biodiversity are ubiquitous under current climate and land use change, and hybridisation has been proposed as either a natural or managed mitigation strategy for the conservation of some susceptible species . Section title: Introduction Educational score: 3.8026928901672363 Domain: biomedical Document type: Study Language: en Although hybridisation can have beneficial outcomes for species or populations via adaptive introgression , it may also lead to reduced biodiversity via species collapse (i.e., fusion of two distinct species or lineages into one), genetic ‘swamping’ (e.g., reduced fitness due to maladaptive hybrids) or even extinction . Therefore, the underlying genetic characteristics and fitness outcomes of hybridisation require study to ensure appropriate management of biodiversity within specific hybridisation contexts. Section title: Introduction Educational score: 4.261707305908203 Domain: biomedical Document type: Study Language: en Within adaptive radiations on islands, introgression among closely related species can be important for the evolution of novel fitness peaks . Species that have undergone relatively recent adaptive radiations often exhibit weak genetic divergence, with phylogenetic lineages best separated with fast‐evolving markers such as microsatellites or single nucleotide polymorphisms (SNPs) , as opposed to more conserved genomic regions . This is particularly relevant for Darwin's finches on the Galapagos Islands in Ecuador, which have reused ancestral modules of genetic variation for beak size over the past million years with introgression, leading to rapid speciation . Further, Darwin's finches are a relatively recent radiation of species with close evolutionary histories, such that previous efforts to untangle the species diversity of finches across islands have employed microsatellites , or whole‐genome phylogenetic analyses . While microsatellites are too sparse and whole‐genome analyses too impractical in large numbers of individuals, ascertaining the incidence of hybridisation in closely related species has been enabled by the generation of dense SNP datasets via high‐throughput sequencing technologies. Section title: Introduction Educational score: 3.893965721130371 Domain: biomedical Document type: Study Language: en Compared to other species groups, the reduced genetic divergence among Darwin's finches has led some to conclude that standard evolutionary paradigms cannot be widely applied to this complex adaptive radiation . Such challenges are similar for the identification of hybrid birds within the group, given the relatively recent divergence of the Darwin's finch assemblage over the past 1.5 million years . Hybridisation and bidirectional introgression among Darwin's finches ( Geospiza spp.) have been well documented on the island Daphne Major, with findings of rare (2%‐5%) but consistent rates of hybridisation across years that are affected by rainfall and resource availability . Section title: Introduction Educational score: 4.0198516845703125 Domain: biomedical Document type: Study Language: en Floreana Island in the Galapagos Archipelago contains two species of Darwin's tree finches ( Camarhynchus spp.) that are undergoing hybridisation, the small tree finch ( C. parvulus ) and the IUCN‐listed Critically Endangered medium tree finch ( C. pauper ) . The common occurrence of hybridisation documented in this population co‐occurred with the confirmed absence of the large tree finch ( C. psittacula ) and hence could have been a result of species collapse . In 2005, the large tree finch was considered rare and possibly absent on Floreana Island , and its presence could no longer be confirmed in 2010 . Instead, there was evidence for one‐way genetic introgression from the extant larger‐bodied finches ( C. pauper ) to smaller‐bodied finches ( C. parvulus ), which was largely driven by C. pauper females pairing with C. parvulus males . The resulting hybrid birds had intermediate body size, and their song was indistinguishable from that of the male parental species ( C. parvulus ) . Kleindorfer et al. used microsatellite data and found a 73% increase in the percentage of Camarhynchus hybrids between 2005 and 2010 following a period of drought and then high rainfall . Section title: Introduction Educational score: 4.193344593048096 Domain: biomedical Document type: Study Language: en Another potential driver of this hybridisation are impacts from the introduced myiasis‐causing ‘avain vampire fly’ Philornis downsi , which causes anaemia, mortality and deformations of the nestling naris via larval blood and tissue parasitism . The number of P. downsi larvae in the nest predicts the size of naris deformation and adult finches with enlarged nares from early‐life parasitism produce low‐quality song that no longer differs between small and medium tree finches . By this mechanism, species recognition may become blurred, and parasitism may promote hybridisation. Notably, hybrid tree finch males on Floreana Island had 50% fewer P. downsi parasites in their nests compared with C. parvulus nests and 60% fewer parasites than C. pauper nests . The effect was also evident according to the inferred degree of Camarhynchus hybridisation, with increased genetic admixture of the attending male correlating with decreased P. downsi within nests . Section title: Introduction Educational score: 4.2311296463012695 Domain: biomedical Document type: Study Language: en This study examines the significance of hybridisation for adaptation by asking: which genes show introgression across species boundaries, and among those genes, which ones show evidence of selection? Given the temporally shifting observation of hybridisation frequency in the Floreana tree finches , this study system offers an opportunity to examine the genomic characteristics of introgression, particularly for loci that may be uniquely selected for advantageous hybrid traits. Such observations may be relevant for understanding the early mechanisms of speciation and evolutionary responses to climate change and parasitism. With a comparison of the Floreana Island Darwin's tree finch population sampled in 2005 and 2013, we aim to: (1) verify previous hybrid genetic assignments based on microsatellite analyses with a new, high‐resolution genome‐wide SNP dataset, (2) examine genome‐wide divergence across the parental and hybrid tree finches and (3) establish whether hybrid tree finches inherit private alleles that are unique to each parental species and whether those alleles shift in frequency across sampling years, show evidence for selection, or map to gene functions relevant for hybrid fitness. Section title: Species Sampling and Study Site Educational score: 3.760831117630005 Domain: biomedical Document type: Study Language: en The study site was in the highlands of Floreana Island (01 17° S, 090 27° W, 300–400 m asl), Galapagos Archipelago, within its humid Scalesia forest at the base of Cerro Pajas volcano. Our study was conducted during the finch breeding season from February to April of 2005 and 2013. The focal tree finch species were small tree finch ( C. parvulus ), medium tree finch ( C. pauper ) and the recently discovered hybrid group that arises from pairings between C. pauper females and C. parvulus males , which all occur in sympatry in the highland habitat dominated by Scalesia pedunculata forest at 400–600 m elevation. Darwin's finches were captured in 6 × 12 m mist‐nets between 600 and 1100 h. Each bird was banded with a numbered aluminium band and a unique combination of colour bands. Morphological measurements and blood samples were taken from adult tree finches as described in Dudaniec et al. and Kleindorfer et al. . Blood samples (10 μL) were immediately transferred to Whatman Classic FTA paper for DNA preservation. All protocols and procedures employed were ethically reviewed and approved by the Flinders University Animal Welfare Committee under permit numbers E190 and E270. Section title: DNA Extraction and RAD Sequencing Educational score: 4.14880895614624 Domain: biomedical Document type: Study Language: en A total of 95 samples of adult tree finches ( Camarhynchus spp.) were selected from samples that were previously identified as parental or hybrid birds using genetic assignment analyses with 10 microsatellite markers . These studies implemented assignment cut‐offs of 0.75 ( C. pauper ) and 0.80 ( C. parvulus ) for parental species assignment using the program STRUCTURE . Based on these prior microsatellite assignments, we extracted DNA from the following individuals. In 2005: 13 C. parvulus , 22 C. pauper and 21 hybrids. In 2013: 12 C. parvulus , 10 C. pauper and 17 hybrids. We extracted DNA from Whatman Classic FTA paper using a modification (200 L volumes used for all washes) of method 4 from Smith and Burgoyne . Two extractions were undertaken per sample, which were then combined, concentrated via evaporation, and quantified on a Qubit 2.0 Fluorometer to obtain 20–40 μL DNA per sample. We constructed Restriction site‐Associated DNA sequencing (RADseq) libraries from the samples using the SbfI restriction enzyme, similar to the method of Baird et al. and following a protocol performed by Floragenex Inc. (Oregon, USA), as described in Text S1 . Samples were paired‐end sequenced on the Illumina HiSeq4000 platform. Section title: Genetic Marker Characterisation Educational score: 4.141183853149414 Domain: biomedical Document type: Study Language: en Raw sequences from each RAD library were quality checked using FASTQC . Each library was demultiplexed using Stacks v2.54 and reads aligned to the Camarhynchus parvulus reference genome STF‐HiC and loci were assembled with gstacks as described in Text S2 and Table S1 . The populations program within Stacks was used to filter SNP loci such that SNPs within a RAD locus had a minimum minor allele frequency (MAF) of 0.03 and were found in a minimum of 75% of individuals within a population. The populations program was run in two stages, (1) with populations defined according to the two parental species and hybrid groups identified from previous microsatellite analyses , and (2) following neutral genetic structure analyses (using the dataset from stage 1), with populations defined according to the revised SNP‐based genetic cluster assignments to either parental species or hybrid group. Section title: Genetic Marker Characterisation Educational score: 4.100068092346191 Domain: biomedical Document type: Study Language: en After stage 1 filtering, there was a total dataset of 10,381 SNPs. A second run of populations was performed with identical parameters but specifying the ‐‐write‐single‐snp flag to retain a single SNP per RAD locus, thus excluding SNPs that are physically linked. This resulted in a SNP dataset of 6637 SNPs for analysis of neutral genetic structure. Individual missingness (i.e., the percentage of missing data per individual specimen) was calculated in vcftools v0.1.13 . For stage 2 filtering (i.e., after undertaking genetic structure analysis—see below), we repeated the above, resulting in 10,463 total SNPs. When using the ‐‐write‐single‐snp flag 6757 SNPs were retained. The stage 2 dataset was used to examine private alleles and genetic divergence within and between each genetic cluster (see below). Section title: Genetic Structure and Hybrid Re‐Identification Educational score: 4.143238067626953 Domain: biomedical Document type: Study Language: en Using 6637 SNPs (from stage 1 filtering), we first assessed pairwise genetic relatedness between our samples to eliminate any full sibs that may be in our dataset. Pairwise relatedness was calculated using the method of Manichaikul et al. implemented in vcftools v0.1.13 . We assessed the microsatellite‐based species and hybrid assignments of our samples by conducting genetic structure analyses using our 6637 SNP dataset. First, a principal component analysis (PCA) was performed using pcadapt in R . The software snmf within the LEA package in R was then used to assign individuals to parental or hybrid groups using individual ancestry coefficients obtained from a sparse nonnegative matrix factorisation ( snmf ) algorithm. The snmf program was run from K 1–10 with 100 repeats per K, iterations were set to 1000 and regularisation parameter α was set to 100. Optimal K was selected based on the lowest cross‐entropy across all runs. Assignment probabilities (i.e., qmatrix values) were used to group individuals into parental (≥ 0.80 q value) or those having hybrid ancestry (< 0.80 q value), in accordance with previous ancestry coefficient thresholds applied to these samples using microsatellites . Section title: Genetic Structure and Hybrid Re‐Identification Educational score: 4.114099025726318 Domain: biomedical Document type: Study Language: en Samples were reassigned as being either hybrid or belonging to the C. parvulus or C. pauper species, according to the results of snmf , prior to subsequent analyses. Notably, we use the term hybrid to describe individuals that show shared ancestry with either parental species based on our assignment threshold (< 0.80 q value), however we were unable to ascertain the number of generations of hybridisation within each sample. Thus, we expected a gradient of assignments to the hybrid group, given previous observations of a hybrid swarm in this system, which likely involves substantial backcrossing between female C. pauper and male C. parvulus or hybrid birds . For visualisation, a second PCA was further performed in pcadapt with reclassified samples. Following this, stage 2 filtering in populations was performed, as described above. Section title: Genetic Structure and Hybrid Re‐Identification Educational score: 4.0146331787109375 Domain: biomedical Document type: Study Language: en Pairwise F ST was calculated between the revised genetic groups using the WC84 estimate implemented in the assigner R package . Bootstrapped 95% confidence intervals (CIs) were calculated using 100 iterations. Observed and expected heterozygosity and F is were calculated for each genetic group (after stage 2) in Stacks . Section title: Genetic Structure and Hybrid Re‐Identification Educational score: 4.169198036193848 Domain: biomedical Document type: Study Language: en To further examine patterns of genetic co‐ancestry among our classified hybrid individuals and parental species, the updated, re‐classified datafile based on snmf assignments was analysed in fineRADstructure in R . This program uses a Markov chain Monte Carlo (MCMC) clustering algorithm to infer population structure and is specifically designed to infer population structure amongst genotypes obtained from RADseq data. First, RAD loci were reordered based on linkage disequilibrium using the script sampleLD, provided within fineRADstructure , followed by calculation of the co‐ancestry matrix with RADpainter . We then ran fineRADstructure with 100,000 burn‐in iterations, followed by 100,000 sample iterations and 1000 iterations for file thinning. FineRADstructure uses haplotype linkage information and focuses on the most recent coalescence (common ancestry) among the sampled individuals to derive a ‘co‐ancestry matrix’—a summary of nearest neighbour haplotype relationships in the dataset. Section title: SNP Outlier Analysis Educational score: 4.299493312835693 Domain: biomedical Document type: Study Language: en We tested for F ST outliers using our stage 2 dataset of SNPs ( N = 10,381; with individuals re‐classified as parentals and hybrids) using three approaches implemented in (1) Bayescan , (2) OutFlank and (3) pcadapt . Bayescan uses a Bayesian framework to detect outliers with high F ST while accounting for sample size , demographic processes and hierarchical genetic structure . Bayescan was run with 50,000 burn‐in iterations, a thinning interval of 10, and sample size of 5000, resulting in 100,000 total iterations. The number of pilot runs was set at 20 with a length of 5000 for each run. We set the prior odds to 10 and implemented a false discovery rate (FDR) threshold of 0.05. Log‐likelihood traces were plotted in R to confirm model convergence. OutFlank is based on the Lewontin and Krakauer method, while accounting for sampling error and nonindependence between sampled populations. OutFlank detects outliers under divergent selection by initially inferring the F ST distribution from multiple loci and then fitting a χ 2 model to the centre of the distribution, resulting in a null distribution. This null distribution is then used to detect outlier loci. We used a left and right trim value of 0.05 as suggested by Whitlock and Lotterhos with K defined by genetic structure analysis described above. Loci with an expected heterozygosity of < 10% were excluded as recommended by Whitlock and Lotterhos , and the FDR was set to 5%. The software pcadapt performs genome scans to detect genes under selection and is effective at handling admixed individuals while not requiring grouping of individuals into populations. Pcadapt uses principal components analysis to account for population structure , and computes p ‐values to identify outlier loci based on the correlation between SNPs and principal components axis (PCs). The numbers of PCs are decided based on a scree plot, which displays the percentage of variance explained by each PC. p ‐Values were adjusted based on the Bonferroni correction with an expected false discovery rate lower than 5%. Section title: Identifying Private Parental Alleles and Divergence in Hybrids Educational score: 4.146877288818359 Domain: biomedical Document type: Study Language: en Using the stage 2 SNPs ( N = 10,381), we calculated per SNP pairwise F ST and Φ ST in Stacks for all loci in both C. parvulus and C. pauper and visualised this across each chromosome of the C. parvulus genome to search for peaks of genetic divergence between parental species . We identified private alleles within each of the parental species (as described in Text S1 ) and mapped them along each of the 30 chromosomes of the C. parvulus genome using the stacks‐private‐alleles program. We further examined the parental private alleles found in the hybrids along each C. parvulus chromosome, according to its frequency in the hybrid samples. We compared private allele frequencies in 2005 and 2013 for both C. parvulus and C. pauper . To identify private alleles that showed increased genetic divergence in the hybrid group, we further identified alleles that occurred in both sampling years and calculated their frequency change across years. Allele frequencies ≥ 0.30 were further identified, which equated to approximately 30% above the mean private allele frequency observed in hybrids. This was examined as a conservative measure of the most confident allele frequency changes in the dataset. To test for an effect of sample size of hybrid individuals across years on, we randomly subsampled the minimum number of hybrids identified in one of the sampled years to establish if differences in sample size affected the patterns in private allele numbers and frequencies observed across 2005–2013. We conducted this subsampling of private alleles three times and averaged values across the subsets for comparison with the original full dataset. Section title: Localised Heterogeneity Across the Genome Educational score: 4.106432914733887 Domain: biomedical Document type: Study Language: en To examine for shifts in localised heterogeneity along chromosomes, which may indicate selection, we conducted local PCA using the program lostruct v.0.9 . Local changes in genetic relatedness across the genome were assessed across each pair of windows using 100 bp windows and 2 PCs. We visualised the resulting dissimilarity matrix across windows using multidimensional scaling (MDS) and variation along the genome was identified by choosing three extreme windows in the MDS plot. We then highlighted these window positions along the C. parvulus genome and created aggregated PCA plots from the extracted corners . Start and end base pair information of each MDS outlier window was extracted. Section title: Combined Evidence for Hybrid Selection Educational score: 4.115189552307129 Domain: biomedical Document type: Study Language: en We integrated the outcomes of the above analyses (i.e., F ST outlier tests, local PCA, private alleles, allele frequencies and changes across years) to find loci that show evidence of selection in hybrids. Specifically, we mapped the 2005 and 2013 allele frequencies of all hybrid private alleles on to the C. parvulus chromosome‐level reference. We subsequently mapped pcadapt outlier loci, and outlier windows from local PCA to identify private alleles or gene regions that correspond with putative outliers and change in frequency across the two sampled years. For private alleles, analyses were done on both the full and subset datasets to establish effects of sample size on the patterns observed. Alleles with multiple lines of evidence for selection were identified as the best candidates. Section title: Gene Annotation Educational score: 4.136523246765137 Domain: biomedical Document type: Study Language: en RAD loci (~400–600 bp in length after assembling paired‐end contigs in Stacks ) that contained 100 bp outlier windows from the local PCA and that contained hybrid private alleles with an allele frequency ≥ 0.3 were identified for gene annotation. Loci containing SNPs identified as outliers via pcadapt were also selected for annotation. These sequences were considered to be candidates under selection and were annotated to the C. parvulus genome using NCBI BLAST with the general nucleotide collection database . E‐value threshold was set to 0.05 and BLASTn was used to match highly similar sequences to the C. parvulus genome. Section title: Genetic Structure and Assignment of Hybrid Ancestry Educational score: 4.120913028717041 Domain: biomedical Document type: Study Language: en None of the samples had missing data > 71% for both the ‘write‐single‐snp’ and the ‘all SNPs’ dataset, thus all 95 samples were included in the analysis (mean missingness = 16%). Pairwise genetic relatedness was low across all samples and no full siblings were detected . Our initial PCA showed two genetic clusters in ordination space, with microsatellite‐analysed hybrid individuals spanning both clusters, with the majority in the C. parvulus cluster . Two individuals appeared as outliers, yet these samples did not have high missing data or high genetic relatedness and were therefore retained in the analysis . The genetic clustering approach of snmf using an 0.80 admixture coefficient ( q ‐value) cut‐off value indicated two genetic clusters and a third admixed group . Section title: Genetic Structure and Assignment of Hybrid Ancestry Educational score: 4.176546096801758 Domain: biomedical Document type: Study Language: en There were notable discrepancies between the prior microsatellite‐based classifications and the present analysis . Of the 38 birds identified to have hybrid ancestry via microsatellite analysis, just seven birds (18%) were assigned less than the 0.80 q ‐value threshold for hybrid assignment using snmf , with the majority being assigned to C. parvulus ( N = 24; 63%) and a few to C. pauper ( N = 4; 11%). This large difference in hybrid assignments was not reflected in the microsatellite‐identified parental species, with 26 of 28 birds (93%) originally assigned to C. parvulus being supported by our SNP data, and the remaining two given newly assigned hybrid status. Similarly, 26 of 32 birds (81%) originally assigned to C. pauper were supported by our SNP data, with six (19%) reassigned hybrid status. This resulted in a revised total of 50 C. parvulus , 30 C. pauper and 15 hybrid birds in our dataset, and all samples were re‐classified accordingly . The mean admixture coefficient ( q ‐value) for birds in (1) the C. parvulus group was 0.915 ± 0.009 s.e, (2) the C. pauper group was 0.946 ± 0.011 s.e and (3) the hybrid group was 0.50 ± 0.052 . The number of previously assigned hybrids decreased by 47.6% in 2005 (from 21 to 11 birds) and 76.5% in 2013 (from 17 to 4 birds) using SNP data . Section title: Genetic Structure and Assignment of Hybrid Ancestry Educational score: 4.234367370605469 Domain: biomedical Document type: Study Language: en The results of our fineRADStructure coancestry matrix showed a clear separation between the two parental species groups, with C. pauper tending to share overall higher ancestry with one another than C. parvulus . The clustering dendrogram formed a total of 15 lineages, with 7 lineages found within the C. parvulus cluster and 8 within the C. pauper cluster. However, some of these lineages (3 within the C. parvulus cluster and 2 from the C. pauper cluster) corresponded with hybrid individuals. Notably, we identified an instance of high co‐ancestry among hybrid_3 and C. parvulus _21 sampled in 2005. The hybrid individual had an ancestry coefficient in snmf of 0.7 to the C. parvulus group, suggesting that this individual may share recent ancestry with C. parvulus _21. Of note is that C. parvulus _21 was also an outlier with respect to having very low genetic relatedness compared to all other samples except hybrid_3 . In addition, individuals C. parvulus _15 and C. parvulus _12 from 2013 showed high ancestry, which is also evident in pairwise relatedness . Although individuals C. parvulus _ 25 and C. parvulus _37 also showed higher than average ancestry , these birds were caught in 2005 and 2013, respectively, therefore recent co‐ancestry is unlikely. Notably, there was a cluster of three hybrid individuals that fell outside of the C. parvulus or C. pauper ‐dominated clusters . Of these, hybrid_12 and hybrid_14 had almost equally shared ancestry coefficients across the two parental clusters (in snmf ) of 0.42 and 0.52 to C. parvulus . All other hybrids had snmf‐ based ancestry coefficients that were between 0.69 and 0.77, and they were clustered within the parental genetic group to which they were more closely assigned in fineRADstructure . Section title: Genetic Structure and Assignment of Hybrid Ancestry Educational score: 3.2428970336914062 Domain: biomedical Document type: Study Language: en Pairwise F ST was highest between the two parental species followed by C. pauper and the hybrid group , with a similarly low F ST between C. parvulus and the hybrid group . This was also reflected in analysis of genetic relatedness between groups, with the genetic relatedness in hybrid birds being intermediate . Section title: Outlier Analysis and Private Alleles Educational score: 4.176007270812988 Domain: biomedical Document type: Study Language: en Both Bayescan and OutFlank F ST outlier tests detected no outlier loci. However, pcadapt detected 69 outlier SNPs across 63 loci that had significantly divergent allele frequencies between the genetic groups identified . Of 10,463 total SNPs analysed, 4869 private alleles were found in the parental species, with the majority being in C. parvulus compared to C. pauper . These private alleles were represented across all 30 chromosomes of the C. parvulus genome . A total of 348 of these parental private alleles had an allele frequency ≥ 0.3 within hybrid birds (mean = 0.43 ± 0.006 s.e, range 0.30–1.00, Table S1 ). Of these high‐frequency private alleles in hybrids, 263 (76%) originated from C. parvulus , and 85 (24%) originated from C. pauper . The majority of the 348 private alleles found in hybrids were at a higher frequency in hybrid birds than in their parental species of origin ( C. parvulus : 253 of 263; 96%; C. pauper : 76 of 85; 89%). This was reduced when considering all private alleles irrespective of their hybrid frequencies, where the allele frequency in hybrids was greater than in C. parvulus for 72% of alleles, and in C. pauper for 43% of private alleles. When restricting comparisons to private alleles that occurred in both sampling years, similar percentages of private alleles were observed from each parental species, with frequencies ≥ 0.3 and with a higher frequency in hybrids (Table S1 ). Section title: Outlier Analysis and Private Alleles Educational score: 4.165134906768799 Domain: biomedical Document type: Study Language: en The effect of sample size on private allele frequencies was assessed using a random subset of four hybrid birds from 2005 , with three subsets performed that were then averaged for comparisons (Table S1 ). Of 10,463 total SNPs analysed, 28% fewer private alleles were found in the parental species, still with the majority being in C. parvulus compared to C. pauper ( N = 747; mean allele frequency = 0.142 ± 0.004 s.e., min = 0.02, max = 0.59, Table S1 ). Compared to the 2005 N = 11 dataset, the subset N = 4 dataset had less than half the percentage of private alleles with a higher frequency in hybrids compared to their species of origin for both species (Table S1 ). However, for the high allele frequency (≥ 0.3) private alleles, the numbers were comparable (143 vs. 191) as well as similar percentages across species (row 6 in Table S1 ). When considering all private alleles irrespective of their hybrid frequencies, the allele frequency in hybrids was greater than in C. parvulus for 73% of alleles, and in C. pauper for 60% (449/747) of private alleles. When reducing the sample size of hybrids to four in 2005 (subsampled data, Table S2 ), the number of private alleles was expectedly fewer, but the percentage of private alleles with a higher frequency in hybrids than parentals was similar in C. parvulus , though less so for C. pauper , and this was not notably different for alleles ≥ 0.3 frequency. Therefore, we did not see markedly different patterns in the patterns of allele frequencies within years using subsampled data as summarised in Table S1 . Section title: Local PCA Educational score: 4.151892185211182 Domain: biomedical Document type: Study Language: en Genome scans using 100 bp windows resulted in 417 windows analysed using a multidimensional scaling (MDS) approach across the C. parvulus genome. An aggregated PCA plot was created from MDS coordinates and two MDS axes were found to significantly explain the variation in the data . The three extreme window clusters identified contained a total of 63 outlier windows . The local PCA outlier windows were mostly randomly distributed across each chromosome of the C. parvulus genome . From these 63 outlier windows, we identified 46 private alleles that were previously identified in hybrid birds with allele frequencies ≥ 0.3. Section title: Private Allele Frequency Shifts in Hybrids Across Years Educational score: 2.7718236446380615 Domain: biomedical Document type: Study Language: en We quantified shifts in private allele frequencies for alleles that occurred in both sampling years. Section title: Private Allele Frequency Shifts in Hybrids Across Years Educational score: 4.167261123657227 Domain: biomedical Document type: Study Language: en Using all samples between 2005 ( N = 11) and 2013 ( N = 4), an allele frequency increase was observed in 278 private alleles originating from C. parvulus (71% of C. parvulus private alleles), and six of these alleles showed a frequency change ≥ 0.3 (mean change all alleles = 0.170 ± 0.002 s.e.; Table S2 ). There were 108 private alleles (76% of C. pauper private alleles) originating from C. pauper and eight of these alleles showed a frequency change ≥ 0.3 (mean change all alleles = 0.113 ± 0.009 s.e.; Table S2 ). A decrease in allele frequency was observed for 79 private alleles (with 8 ≥ 0.3) originating from C. parvulus (20% of C. parvulus private alleles; mean change all alleles = 0.129 ± 0.011 s.e.), which was reduced in C. pauper private alleles (19 alleles, 13% of C. pauper private alleles; 2 ≥ 0.3; mean change all alleles = 0.106 ± 0.019 s.e; Table S2 ). Therefore, C. parvulus is contributing more private alleles to hybrids overall, and the majority of private alleles show an increase in frequency irrespective of species of origin. Section title: Private Allele Frequency Shifts in Hybrids Across Years Educational score: 4.104052543640137 Domain: biomedical Document type: Study Language: en To examine for impacts of hybrid sample sizes, we recalculated the above patterns across years for the averaged subset data and compared them to the 2005 N = 11 dataset (Table S3 ). While the number of private alleles identified to have shifted was greatly reduced overall, so was the percentage of alleles that increased in frequency 2005–2013 in the subset data, irrespective of species of origin ( C. parvulus : 11% of 149 private alleles, 0.7 ≥ 0.3; C. pauper : 19% of 36 private alleles, 2 ≥ 0.3; Table S2 ). A similar, but opposite discrepancy was found for private alleles decreasing in allele frequency, with higher percentages of alleles decreasing across both species ( C. parvulus : 61% of 149 private alleles, 22 ≥ 0.3; C. pauper : 61% of 36 private alleles, 5 ≥ 0.3). Overall, the differences between the original and subset data were substantial with respect to detecting shifts in private allele frequencies between 2005 and 2013, indicating that sample size effects are present for these comparisons (Table S3 ). Section title: Private Allele Frequency Shifts in Hybrids Across Years Educational score: 4.139040946960449 Domain: biomedical Document type: Study Language: en We next examined the correspondence between private alleles , pcadapt outlier loci, and local PCA outlier windows. A total of 46 pcadapt outliers mapped to the chromosome level genome assembly for C. parvulus , and of these, 29 outliers aligned directly with a private parental allele found within the hybrid samples . These outlier private alleles each occurred on a separate chromosome so were physically unlinked and were distributed across 21 of the 30 C. parvulus chromosomes, with 1–6 outlier alleles per chromosome . Of these 29 private outlier alleles, 28 showed allele frequency changes from 2005 to 2010, with 11 C. parvulus and three C. pauper alleles increasing in allele frequency. In contrast, 13 private outlier alleles from C. parvulus and one allele from C. pauper decreased in allele frequency between 2005 and 2013 , however we urge caution regarding allele frequency changes given the large sample size effect on private allele frequencies established above (Table S3 ). Section title: Gene Annotation Educational score: 4.149394989013672 Domain: biomedical Document type: Study Language: en A total of 59 unique gene annotations were obtained from the 63 outlier sequences identified from pcadapt (Table S2 ). From the loci containing the 46 hybrid private alleles from the MDS outlier windows, 22 uniquely annotated genes were identified (Tables S4 and S5 ). Across all annotations, 14 loci were annotated to 2–7 variants of the same gene (Table S4 ). All annotations were found to be protein‐coding genes or gene variants and had an average percentage match identity of 89.9% ( n = 125 total annotations including variants; range: 77.9%–100%). All 14 overlapping annotations were from the same locus on chromosome 17 . The remaining seven loci were annotated to chromosome 4A ( n = 1), chromosome 5 ( n = 2), chromosome 26 ( n = 1) and chromosome 28 ( n = 2) and the Z chromosome ( n = 1). Section title: Gene Annotation Educational score: 4.101559638977051 Domain: biomedical Document type: Study Language: en Many gene annotations were associated with inflammation and immunity. For example, one private outlier locus was found within an MDS outlier window, could be annotated to the C. parvulus gene leucine‐rich repeat containing 40 . Within the 22 annotated outliers were other genes involved in inflammation and immunity (e.g., ankyrin repeat and SOCS box containing 12 [ASB12], E3 ubiquitin protein ligase 2 [SIAH2], pentraxin 3 [PTX3], and growth differentiation factor 15 [GDF15]), brain function , thrombospondin type laminin G domain, and EAR repeats (TSPEAR), UTP15 small subunit processome component (UTP15), as detailed further in Tables S4 and S5 . Section title: Discussion Educational score: 4.34101676940918 Domain: biomedical Document type: Study Language: en We provide evidence that genomic introgression among Darwin's tree finches, the Critically Endangered C. pauper and stable C. parvulus , introduces high‐frequency private alleles in to hybrids that selection may act on across years. Alleles that were unique to each parental species appeared in hybrid birds at frequencies higher than within their parental species of origin. Many of these alleles were identified as candidate outliers under selection or appeared in divergent genomic windows . We also identified a wide range of changes in hybrid private allele frequencies over an 8‐year period, and this was sensitive to the sample size of birds. Some private alleles were located within genes that code for functions that may be relevant for hybrid fitness. Therefore, the retention of genetic diversity via hybridisation in this population of Camarhynchus may be beneficial for the adaptive capacity of C. pauper , which is under increasing threat from local extinction due to habitat degradation and P. downsi . Our findings further elucidate the limitations of microsatellite‐based hybrid assignment in closely related and sympatric species with greater certainty afforded by high resolution, genome‐wide SNP markers. Darwin's finches offer an unparalleled opportunity to study the earliest stages of species hybridisation in a vertebrate , and here they reveal the role of introgression for maintaining neutral and adaptive genetic variation of endangered species. Section title: Mismatches in Hybrid Identification Educational score: 4.211607456207275 Domain: biomedical Document type: Study Language: en Our results suggest that the number of hybrid tree finches on Floreana is overestimated using microsatellite markers . We document a much lower hybrid assignment rate using SNPs yet confirm a swarm of backcrossed C. parvulus and hybrid finches, which presents a challenge for classifying individuals based on genetic clustering approaches. Notably, our results do not provide further insight into the shift in the frequency of hybridisation across years that was previously documented in this population because we analysed a nonrandom sample of birds that was preselected based on prior microsatellite assignments. Furthermore, this study does not negate the findings of female‐biased introgression from C. pauper to C. parvulus in Peters et al. , as their conclusions were based on genetic assignment probabilities to each parental species rather than distinct groups of hybrids and parental individuals. We found that the number of hybrid birds assigned using SNP data was 47.6% lower in our 2005 sample (from 21 to 11 birds) and 76.5% lower in our 2013 sample (from 17 to 4 birds) compared to the initial microsatellite‐based assignments . Notably, the co‐ancestry approach of fineRADstructure grouped just three hybrid birds outside of the two parental clusters, and these all had ~0.5 assignment probability to each parent, indicating potential first‐generation hybrids without backcrossing . The rest of the hybrid‐assigned birds had snmf assignment probabilities to each parental cluster that were lower or higher than 0.5. The detection of birds with hybrid ancestry in fineRADStructure therefore appears to be conservative, perhaps influenced by the recent ancestry of the parental species. Of note is that in the current study we use a different genetic clustering approach implemented in the R package snmf , while previous studies used the program Structure , though we use the same assignment cut‐off value of 0.80 in this study as in Peters et al. . This difference in program usage is not likely to have resulted in the large differences we report in hybrid assignments between the SNP and microsatellite analyses, particularly as we used three approaches to determine hybrid status (e.g., PCA, snmf and fineRADStructure ). Section title: Mismatches in Hybrid Identification Educational score: 4.11386251449585 Domain: biomedical Document type: Study Language: en For genetic structure analyses, previous studies of threatened species that have compared SNP and microsatellite data have found similar patterns, but SNP data often provide stronger evidence of hierarchical genetic structure, and increased evidence for population evolutionary independence , with genetic assignments sometimes varying across lineages . Marker comparisons for detecting hybrid animals have found microsatellites to overestimate hybridisation frequencies compared with high‐resolution SNP markers that have a much higher density and wider coverage of the genome . SNPs may also reveal higher resolution of backcrossed individuals that were missed by microsatellites . Overall, this suggests that SNP approaches outperform microsatellite‐based assessments of hybridisation frequency. Section title: Mismatches in Hybrid Identification Educational score: 4.123325824737549 Domain: biomedical Document type: Study Language: en Our analyses support the ‘hybrid swarm’ scenario observed by Peters et al. , whereby a continuum of hybrid ancestry exists between the two parental tree finch species, though we find a reduced proportion of hybrids in our dataset than previously reported . This suggests that there are fewer tree finch hybrids in the Floreana population than previously thought, which may be promising for maintaining the genetic integrity of endangered C. pauper . Notably, hybrid recruitment in the tree finch population on Floreana increases in years with higher rainfall , thus additional sampling periods would enable the impact of climate fluctuations to be accounted for in estimates of hybridisation frequency. Although we advance present understanding using a RADseq approach, our study could be further improved with a whole‐genome sequencing approach that enables a higher density genome scan of diversity and selection, as well as structural variants that may be under selection in hybrids. Section title: Private Alleles and Evidence for Selection in Hybrids Educational score: 4.3866448402404785 Domain: biomedical Document type: Study Language: en Theory predicts that genome‐wide patterns of introgression in hybridising populations will vary and are influenced by either population size variation or selection . We find widespread evidence for introgression across the genome (using the C. parvulus reference genome), with parental private alleles in hybrids, outlier loci and divergent genome windows located on nearly all 30 C. parvulus chromosomes. As these loci were not clustered within specific genomic regions, it is unlikely that we have missed hotspots of introgression across the finch genome. Most of the 348 high‐frequency (≥ 0.30) private alleles found in hybrids were at a lower frequency in their parental species of origin, indicating that either genetic drift or selection in hybrids is driving this difference. With 90% of private hybrid alleles (67% C. parvulus , 23% C. pauper ; Table S2 ) being retained across years and undergoing allele frequency change over the 8‐year sampling interval, a case for selection is strengthened alongside the outlier loci and local PCA outlier windows that overlap with these private alleles . Notably, 30% of the private alleles showed a decrease and 70% an increase in frequency in hybrids between 2005 and 2013 (Table S3 ), however, these percentages differed markedly when we subset the data in 2005, with more alleles decreasing (61%) than increasing (30%) in frequency. The larger percentages of decreasing private allele frequencies in the 2005 subset data may be due to there being falsely higher allele frequencies due to reduced sample size, resulting in an inflation in the number of alleles found to decrease in frequency in 2013. Therefore, we urge caution in the interpretation of specific allelic shifts yet maintain that our estimates offer a plausible range of frequency shifts in private hybrid alleles. These shifts may be due to genetic drift or selection processes, but a larger sample size, ideally over a longer period would be needed to address this. Section title: Private Alleles and Evidence for Selection in Hybrids Educational score: 4.170404434204102 Domain: biomedical Document type: Study Language: en Given the cryptic hybridisation found in this group of birds, as in many other species groups, this study will aid future work in identifying hybrids based on the presence of private alleles. Further, through the identification of private alleles, this study will aid in identifying genetically ‘pure’ members of the endangered C. pauper on Floreana and help to understand positive or negative roles that hybridisation may play for this tree finch population. Indeed, hybridisation is increasingly heralded as a conservation tool rather than a hindrance . Accordingly, we previously found that nests of hybrid Camarhynchus birds on Floreana harbour fewer parasites of the avian vampire fly, P. downsi , compared to nests of parental species , suggesting that hybridisation confers some resistance to P. downsi , which in turn may favour greater genetic introgression due to heterosis . The increasing numbers and co‐evolutionary shifts of P. downsi in Floreana finch nests have been well documented over the past two decades , alongside rates of tree finch hybridisation . Further, hybrid birds are reported to be much more common on Floreana Island than on the island of Daphne Major, where P. downsi is in low numbers, and hybrids occur in just 2%–5% of breeding pairs . Section title: Private Alleles and Evidence for Selection in Hybrids Educational score: 4.309990882873535 Domain: biomedical Document type: Study Language: en As introgression should favour alleles beneficial for hybrid fitness, the ancestry of beneficial genotypes within hybrids is expected to show an excess of ancestry towards the introgressing species . In Darwin's tree finches on Floreana Island, the more common and stable C. parvulus is contributing more of its genetic material to hybrid birds than the critically endangered C. pauper via backcrossing from hybrids to C. parvulus . Our findings support this, with C. parvulus private alleles being more common in hybrids . These C. parvulus private alleles were also more likely to show substantial allele frequency change across the 8‐year sampling period . Thus, the patterns of introgression we observe indicate variation in retention or selection for private alleles originating from C. parvulus , while alleles from C. pauper remain more stable. Several striking examples demonstrate adaptive introgression during invasions of novel species into a location, or for congeneric species in sympatry or allopatry. For example, Valencia‐Montoya et al. discovered the transfer of pesticide resistance alleles from invasive moths into local native moths via genomic introgression. However, introgression can have positive, negative or neutral effects and evolutionary forces acting within hybrid populations may effectively ‘filter’ gene flow between species . Introgressed DNA is typically thought to be deleterious in the recipient species , therefore investigation into the incidence of purging of deleterious alleles over our study period may further elucidate the genetic mechanisms underlying this hybridisation. Section title: Gene Annotations to Outlier Loci and Private Alleles Educational score: 4.465470314025879 Domain: biomedical Document type: Study Language: en When selection acts on hybridising species, genes linked to reproductive isolation tend to resist introgression, while alleles that confer locally adaptive traits do not . Here we found candidate loci under selection containing private alleles in hybrid tree finches that also mapped to genes associated with inflammation and immunity, brain function and development (Table S3 ). Notably, the gene for leucine‐rich repeat containing 40 (LRRC40) was an outlier in pcadapt , found within an MDS outlier on chromosome 5 and increased in frequency by 0.5 between 2005 and 2013 (Table S4 ). This gene is involved in signal transduction and has been linked with autism in humans , and such leucine repeats are found in many proteins associated with innate immunity in vertebrates . The annotated gene DTX4 is involved in notch signalling and is part of the pathway network for cytosolic sensing of pathogen‐associated DNA, and the innate immune system . Notably, DTX4 was found to be differentially expressed in urban and non‐urban common kestrels . Other genes of note that had lower query cover and percentage identification included E3 ubiquitin protein ligase 2 (SIAH2), which is known to suppress growth of avian reovirus, an important virus of chickens . Pentraxin 3 (PTX3) is involved in the anti‐inflammatory immune response and may be used as an acute phase protein marker to monitor inflammatory conditions in poultry . Ninjurin 1 has well‐known nerve regenerative and anti‐inflammatory effects but is not studied in birds . For genes involved in cognition, the acid‐sensing ion channel is involved in detecting extracellular acidification in the brain and aids in both chemosensation and mechanosensation for monitoring homeostasis and pathological signals . The gene for solute carrier family 7 member 5 (SLC7A5) has been widely studied in chickens in the context of nutrient transport, metabolism and digestion , but the gene has also been associated with feather pigmentation and follicle development in ducks . In development, the gene EBF2 transcription factor is involved in ligament formation and limb development in chickens . These gene annotations to private alleles in hybrids that are also candidate loci under selection suggest that introgression among Camarhynchus confers functional genetic effects that may be further investigated for fitness correlates. Section title: Conclusion Educational score: 4.120797157287598 Domain: biomedical Document type: Study Language: en Our study provides evidence that natural hybridisation can provide important genetic variation that impacts both ecological and evolutionary processes. We document frequent introgression in Darwin's tree finches on Floreana Island that is promoting gene flow and novel selection pathways in hybrids that may be influencing the evolutionary trajectory of Camarhynchus on the Galapagos Islands. Notably, the island of Floreana is undergoing a massive ecological restoration effort involving avian predator removal and species re‐introductions, which may shift selection pressures and hybridisation frequencies for Camarynchus going forward. Our study therefore has implications for monitoring the genetic diversity and integrity of the Critically Endangered medium tree finch ( C. pauper ) as Floreana transitions into a state of ecological restoration. Section title: Conflicts of Interest Educational score: 0.8662789463996887 Domain: other Document type: Other Language: en The authors declare no conflicts of interest. | Review | biomedical | en | 0.999997 |
PMC11695275 | Section title: Introduction Educational score: 4.1421613693237305 Domain: biomedical Document type: Review Language: en Ankylosing spondylitis (AS) is a chronic, progressive inflammatory disease involving the spine and sacroiliac joints ( 1 , 2 ). The prevalence of AS varies significantly across different regions, ranging from 0.74% in Africa to 3.19% in North America ( 3 ). In addition to inflaming the sacroiliac joints and spine, AS can cause extra-articular symptoms such as anterior psoriasis, uveitis, or inflammatory bowel disease ( 4 ). In severe stages of the disease, persistent inflammation can cause fibrosis and calcification of spinal joints, restricting joint mobility and potentially leading to joint fusion, which may ultimately result in disability ( 5 ). Due to the insidious onset and subtle early symptoms of AS, its diagnosis is often delayed. For instance, in the United States, the average time from symptom onset to referral is approximately one year, whereas in Western Europe and other regions, this time can exceed three years, frequently leading to missed treatment opportunities and delayed therapy ( 6 ). Section title: Introduction Educational score: 4.029547691345215 Domain: biomedical Document type: Review Language: en Although the exact cause of AS is still unknown, environmental and genetic factors such as autophagy, inflammatory cytokines, certain bacterial infections, and macrophage activation are thought to have a role in its pathogenesis ( 7 ). Due to a strong familial predisposition of AS, early research highlighted the significance of genetic factors in its pathogenesis. However, as our understanding of the disease has deepened, it has become evident that the currently used biomarkers, such as HLA-B27 status, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR), provide only moderate diagnostic and prognostic utility. There is a pressing need for improved biomarkers in AS to facilitate early diagnosis, improve prediction of therapeutic responses, and facilitate the assessment of long-term outcomes in AS. Recent advancements in transcriptomics technologies and statistical methodologies offer promising opportunities to identify and develop more informative biomarkers for such clinical applications ( 8 , 9 ). Section title: Introduction Educational score: 4.181556224822998 Domain: biomedical Document type: Study Language: en RNA sequencing (RNA-seq) has become a novel high-throughput sequencing technique in recent decades. It is capable of recognizing abnormally spliced genes, detecting allele-specific expression, and identifying differentially expressed genes (DEGs) ( 10 ). Bioinformatics analysis has been utilized to elucidate abnormal biological processes underlying disease pathogenesis and can leverage sequencing data to assess an organism’s genome, transcriptome, and proteome information ( 11 ). To date, several studies have identified DEGs implicated in the pathogenesis of AS using microarray and RNA-seq techniques. Peripheral blood is widely recognized as a promising resource for identifying transcriptomic biomarkers ( 12 ). Notably, peripheral blood biomarkers have achieved significant success in predicting tumor onset and progression, such as in lung and breast cancers, as well as in the detection and drug development of Alzheimer’s disease ( 13 – 15 ). Nevertheless, the DEG levels in the peripheral blood of AS patients have yet to be fully elucidated, and the aforementioned molecular mechanisms remain to be further validated. Section title: Introduction Educational score: 4.265511989593506 Domain: biomedical Document type: Study Language: en In this study, gene microarray expression data from GSE73754 and GSE25101 were obtained from the GEO database. Using bioinformatics analysis, DEGs in the serum of patients with AS and normal controls were identified. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were performed to explore their functions and pathways. Then, key genes were identified, and further receiver operating characteristic (ROC) analysis of these key genes was conducted. This study identified a strong association between the key genes and ferroptosis, a newly recognized form of programmed cell death with a critical role in various inflammatory diseases. In patients with ankylosing spondylitis (AS), abnormalities in iron metabolism and oxidative stress are hallmark pathological features, suggesting that ferroptosis may significantly contribute to the development and progression of AS ( 16 ). Furthermore, through immune infiltration analysis, this study explored the disease microenvironment of AS, uncovering the potential involvement of immune cells in its pathogenesis. As AS is a chronic inflammatory disease characterized by immune abnormalities, the findings on immune infiltration offer deeper insights into its immunopathological mechanisms ( 2 ). Finally, the expression levels of key genes and their correlation with various clinical indicators were validated by real-time quantitative PCR. In addition to offering fresh information on the pathological mechanisms of AS, the study suggested new potential biomarkers and targets for AS diagnosis and treatment. Section title: Data collection Educational score: 3.938528060913086 Domain: biomedical Document type: Study Language: en Gene expression data GSE73754 and GSE25101 were derived from the GEO database ( 17 , 18 ). Based on the GPL10558 platform, the GSE73754 dataset included 72 samples, of which 52 were peripheral blood samples from patients with AS while 20 were from healthy controls. Based on the GPL6947 platform, the GSE25101 dataset included 32 samples, comprising peripheral blood samples from 16 patients with AS and 16 healthy controls. Section title: Differential expression analysis and enrichment analysis Educational score: 4.096446990966797 Domain: biomedical Document type: Study Language: en In the GSE25101 and GSE73754 datasets, DEGs were screened. DEGs were identified using the Limma R package, with a significance threshold defined when the P -value was less than 0.05 and the FoldChange was greater than 1.2 ( 19 ). The R packages “ggplot2” and “pheatmap” were utilized to visually present the DEGs via volcano plots and heatmaps ( 19 ). GO and KEGG functional enrichment analyses were executed via the “clusterProfiler” and “enrichplot” R packages. Additionally, GSEA was conducted based on GSE73754 using the “clusterProfiler” and “ReactomePA” R packages to identify relevant enriched signaling pathways ( 20 , 21 ). Section title: Weighted gene co-expression network construction and module analysis Educational score: 4.124311923980713 Domain: biomedical Document type: Study Language: en Each gene’s median absolute deviation (MAD) was determined, and the top 50% of genes were chosen based on their MAD values. To examine the connection between co-expressed genes and phenotypes, a gene co-expression network was built ( 22 ). Gene comparison was done via average linkage methods and Pearson correlation matrices. By utilizing the weighted adjacency matrix and the soft-thresholding parameter β, a scale-free co-expression network was established. The adjacency matrix was raised to the power of 14 to convert into a topological overlap matrix (TOM), which was used to gauge the network connectivity of genes. With the average linkage hierarchical clustering and TOM-based dissimilarity measures, the correlation among modules was identified, with the minimum gene module size set to 10. Section title: Core gene selection and logistic regression model construction Educational score: 4.08154821395874 Domain: biomedical Document type: Study Language: en The intersection of co-expressed DEGs from GSE73754 and GSE25101 datasets with WGCNA module genes identified 6 genes. LASSO regression was then adopted to simplify the model, identifying 5 key genes, which were utilized to establish a diagnostic model for AS ( 23 ). The diagnostic performance of key genes and the logistic regression model were evaluated using the R package “ROCR”. Subsequently, a nomogram for predicting AS risk based on characteristic genes was constructed using the R package “rms”, and its predictive efficacy was estimated through calibration curves. Section title: Immune analysis algorithm Educational score: 4.0620436668396 Domain: biomedical Document type: Study Language: en Based on the expression levels of genes relevant to immune cells, the ssGSEA algorithm was adopted to determine the infiltration levels of different immune cells. An immune cell composition matrix for analysis was created by integrating the output data for 28 different categories of immune cells. The correlation between core biomarkers and immune infiltrating cell expression was analyzed using non-parametric Spearman correlation. The “corrplot” R package was then employed to draw correlation heatmaps. Section title: Study procedure Educational score: 4.0398125648498535 Domain: biomedical Document type: Study Language: en From Mar. 2024 to Jun. 2024, 24 drug-naive patients with AS meeting the modified New York diagnostic criteria were selected from the Affiliated Hospital of North Sichuan Medical College, and blood samples were collected from 24 healthy male volunteers (healthy control group, HC group) ( 24 ). All participants had no history of cardiovascular disease, diabetes mellitus, hepatitis, malignancies, or other autoimmune and inflammatory diseases. The study was approved by the Affiliated Hospital of North Sichuan Medical College’s Ethics Committee, with informed consent obtained from all participants . 4 mL of heparin-anticoagulated peripheral venous blood was used for the isolation of peripheral blood mononuclear cells (PBMCs). The Trizol technique was utilized to extract total RNA. Gene primers were designed as per the gene sequences of ACSL1, SLC40A1, GZMM, TRIB1, and XBP1 in PubMed Gene and synthesized by Sangon Biotech (Shanghai) Co., Ltd. ( Table 1 ). Section title: Statistical analysis Educational score: 3.8232805728912354 Domain: biomedical Document type: Study Language: en All bioinformatics statistical analyses and visualizations were performed using R (version 4.3.2). Values from the experiment were reported as mean ± standard deviation (SD). In addition, to verify the data normality, the Shapiro-Wilk test was adopted. The t-test or the Mann-Whitney U test was adopted for the analysis based upon whether the data set exhibited a normal distribution, while Pearson or Spearman tests were utilized for correlation analysis. All experimental statistical analyses were conducted using SPSS v27 or GraphPad Prism (v10.2.3). At least three independent replications of each experiment were conducted. P < 0.05 was used to define significance. Section title: DEG identification in ankylosing spondylitis Educational score: 4.092838287353516 Domain: biomedical Document type: Study Language: en The flowchart of this study is illustrated in Figure 1 . We investigated high-throughput sequencing data from the GSE73754 and GSE25101 databases, pertaining to patients with AS and healthy controls. A total of 196 DEGs were successfully identified from the GSE73754 dataset, including 121 upregulated genes and 75 downregulated genes. From the GSE25101 dataset, 576 DEGs were identified, comprising 297 upregulated genes and 279 downregulated genes. These DEGs were visualized using volcano plots , and the top 100 DEGs were presented in heatmaps . Based on the differential gene expression trends from these two datasets, 9 co-expressed DEGs were obtained, with 5 upregulated genes and 4 downregulated genes . Section title: Potential function and pathway analysis Educational score: 4.480556964874268 Domain: biomedical Document type: Study Language: en GSEA analysis of the GSE73754 dataset showed significant downregulation of rRNA processing and translation processes , indicating that these key biological processes were potentially suppressed under the studied conditions. Additionally, osteoclast differentiation-, Streptococcus infection-, and tuberculosis-related genes were significantly upregulated, while oxidative phosphorylation process-related genes were downregulated , suggesting changes in immune response and metabolic activity in AS. GO and KEGG enrichment analyses were performed on the 9 co-expressed DEGs obtained by integrating the GSE73754 and GSE25101 datasets. GO analysis identified 188 significantly different GO terms, including 162 biological processes, 3 cellular components, as well as 23 molecular functions. These genes were enriched in key biological processes such as leukocyte-mediated immunity and positive regulation of angiogenesis. Regarding the cellular component, the peroxisomal membrane and phagocytic vesicle membrane were highlighted. In terms of the molecular function, growth factor binding and protein kinase regulator activity pathways were enriched . These genes are mainly involved in immune response, cytotoxicity, and regulatory processes, indicating a close association between AS and abnormal immune responses and cellular regulatory mechanisms. KEGG analysis unraveled that 11 pathways were enriched, including ferroptosis, fatty acid metabolism, mineral absorption, as well as Th1, Th2, and Th17 cell differentiation . The enrichment of these pathways indicated substantial alterations in immune response, metabolic activity, and cell death mechanisms in AS, offering crucial insights into the underlying biological processes. Section title: Construction of weighted gene co-expression network Educational score: 4.101458549499512 Domain: biomedical Document type: Study Language: en By WGCNA, co-expressed gene clusters with differential expression in the GSE73754 dataset were identified, and the relationship between combined modules and disease traits was calculated . The soft-threshold power was set to 15 , and 4 modules were identified . The grey module showed the most robust positive correlation with the occurrence of AS (r = 0.48), and 121 genes from the grey module were screened . Section title: Validation of the diagnostic model based on key genes Educational score: 4.171283721923828 Domain: biomedical Document type: Study Language: en The intersection of the 9 co-expressed DEGs from the GSE73754 and GSE25101 datasets with the 121 grey module genes identified by WGCNA resulted in 6 core genes . Using LASSO regression to simplify the model , 5 key genes (ACSL1, SLC40A1, GZMM, TRIB1, and XBP1) were identified. Box plots were then adopted to show the expression trends of these 5 key genes in the GSE73754 and GSE25101 datasets . Subsequently, ROC analysis was made to evaluate the potential of these 5 key genes as diagnostic biomarkers for AS . All key genes had an AUC greater than 0.7, indicating favorable clinical diagnostic performance. A nomogram predicting AS risk based on these 5 key genes was constructed , with each gene corresponding to a scoring standard. The calibration curve attested to the favorable predictive performance of the model . Additionally, the ROC curve analysis showed that the overall AUC of the model was 0.807 , indicating that the core genes have high diagnostic performance. Section title: Correlation analysis concerning immune cell infiltration and key genes Educational score: 4.132104873657227 Domain: biomedical Document type: Study Language: en An analysis of the infiltration of 28 immune cell subtypes between the AS group and the control group revealed that six immune cell subsets showed statistically significant differences . In the AS group, central memory CD8 T cells and neutrophils were increased, while activated CD8 T cells, activated dendritic cells, type 1 helper T cells, and γδT cells were decreased . Furthermore, the correlation analysis between key genes and the aforementioned differential immune cell subsets showed that ACSL1, SLC40A1, and TRIB1 were positively linked to neutrophil infiltration, whereas GZMM and XBP1 were negatively linked to neutrophil infiltration. GZMM and XBP1 were positively linked to γδT cell infiltration, while ACSL1 was negatively linked to γδT cell infiltration. TRIB1 was positively linked to central memory CD8 T cell infiltration. GZMM and XBP1 were positively linked to activated CD8 T cell infiltration, while ACSL1, SLC40A1, and TRIB1 were negatively linked to activated CD8 T cell infiltration . Section title: RT-qPCR expression and clinical correlation Educational score: 4.100291728973389 Domain: biomedical Document type: Study Language: en To validate the role of key genes, an RT-qPCR assay of the mRNA expression levels of the five key genes in PBMCs from AS patients and healthy individuals was performed . The results showed that, compared to healthy controls, the expression level of ACSL1 and SLC40A1 was upregulated in AS, while GZMM and XBP1 expression was downregulated, consistent with the expression trends predicted by the model. However, TRIB1 expression was downregulated, contrary to predicted expression by the model . Correlation analysis between the five key genes and clinical indicators (including blood analysis, hsCRP, ESR, HLA-B27, gender, and age) ( Table 2 ) showed that ACSL1 was positively linked to patient hsCRP levels . GZMM was negatively linked to patient ESR levels , hsCRP levels , and neutrophil count . SLC40A1 was positively linked to patient ESR levels (r = 0.54, P < 0.01), hsCRP levels , and neutrophil count . XBP1 was negatively linked to patient ESR levels , hsCRP levels , and neutrophil count . Section title: Discussion Educational score: 4.084404468536377 Domain: biomedical Document type: Study Language: en This study identified five key genes (ACSL1, SLC40A1, GZMM, TRIB1, and XBP1) as potential biomarkers for AS by analyzing the GSE73754 and GSE25101 datasets from the GEO database. The mRNA expression levels of these key genes in PBMCs from AS patients and healthy individuals were validated using RT-qPCR assay. Furthermore, these genes may be linked to disease activity and may be useful in the diagnosis of AS, according to the correlation analysis between the key gene expression and clinical data. Section title: Discussion Educational score: 4.3408026695251465 Domain: biomedical Document type: Study Language: en AS typically leads to calcification and bone formation, accompanied by destructive bone lesions. New bone formation within the axial skeleton is a characteristic of post-inflammatory AS ( 25 ). Innate cytokines, specifically the interleukin-23/17 axis, have been shown in recent research to have a critical role in the pathogenesis of AS ( 26 , 27 ). Clinically, anti-IL-17 therapy has been proven effective in improving bone destruction in AS, but some patients do not respond to IL-17 treatment ( 28 ). Our study found that the IL-17 pathway was also enriched in the co-expressed trend genes identified, consistent with current research. Additionally, KEGG analysis revealed that were adipocytokine signaling pathway, mineral absorption pathway, PPAR signaling pathway, and Th1 and Th2 cell differentiation pathways were enriched, all of which have been reported to have significant value in bone metabolism research ( 29 – 32 ). The study also identified several genes related to bone metabolism and osteocyte function. GO analysis revealed multiple biological processes related to osteocyte differentiation and bone metabolism, such as bone mineralization, osteoblast differentiation, and osteoclast differentiation. GSEA uncovered key pathways, including osteoclast differentiation, suggesting the active role of osteoclasts in AS. Additionally, GSEA revealed metabolic pathways such as oxidative phosphorylation. These pathways are closely related to osteoclast function and bone metabolism. Section title: Discussion Educational score: 4.465463638305664 Domain: biomedical Document type: Study Language: en Among the core genes in this study, ACSL1 is a key enzyme in fatty acid metabolism, mainly responsible for converting long-chain fatty acids into acyl-CoA, a prerequisite for fatty acids to participate in metabolic pathways such as β-oxidation and lipid synthesis ( 33 ). Studies have shown that lipid molecules containing 18:3 chains significantly decline in cells lacking ACSL1, while the presence of ACSL1 increases the synthesis and accumulation of these lipids, which may promote ferroptosis through oxidation ( 34 – 36 ). The experiment results demonstrated that ACSL1 was upregulated in AS patients and exhibited a positive correlation with hsCRP levels. This suggested that ACSL1 may promote ferroptosis through fatty acid metabolism, thereby enhancing the inflammatory response in AS. SLC40A1 is an iron transporter protein responsible for transporting intracellular iron to the extracellular space, playing an important role in iron metabolism. SLC40A1 influences the iron content and function of immune cells such as dendritic cells by regulating iron export, thereby modulating immune responses ( 37 , 38 ). According to the findings of our investigation, AS patients had upregulated SLC40A1, which positively linked to both hsCRP and ESR. This finding suggests that it may indirectly enhance the inflammatory response in AS by affecting the iron content of immune cells like dendritic cells. As a serine protease belonging to the granzyme family, GZMM is mostly released by cytotoxic lymphocytes, including CD8 + T cells and natural killer cells. It plays a role in immune defense and target cell lysis ( 39 , 40 ). In this study, GZMM was downregulated in patients with AS and negatively linked to multiple immune cell infiltrations, suggesting that GZMM may inhibit inflammation by modulating the activity of neutrophils and γδT cells. Notwithstanding its lack of catalytic activity, the pseudokinase TRIB1 is crucial in cell signaling and gene expression regulation. It has an impact on various cellular processes, such as metabolism, inflammation, and cell differentiation ( 41 ). In this study, the RT-qPCR expression of TRIB1 in AS patients contradicted the predictive model, which may be attributable to various factors. The model’s prediction of elevated TRIB1 expression might reflect its role in diverse and complex cellular signaling pathways. In contrast, the lower RT-qPCR expression may result from changes in the proportion of specific cell subsets within PBMCs of AS patients, potentially affecting overall expression levels. Furthermore, TRIB1 expression could be dynamically regulated by microenvironmental factors, such as inflammatory cytokines and oxidative stress, contributing to the observed discrepancies between the model and experimental data. Moreover, the predictive model, which relies on big data statistical analysis, may be influenced by sample heterogeneity and data normalization methods. In comparison, RT-qPCR results depend on experimental conditions (e.g., RNA quality, primer design), with these technical differences possibly accounting for the observed inconsistencies. In this study, TRIB1 was downregulated in AS patients and positively correlated with neutrophil and central memory CD8 T cell infiltration, suggesting that TRIB1 may play a key role in regulating neutrophil activity and quantity as well as maintaining CD8 T cell stability. XBP1 is an important transcription factor involved in endoplasmic reticulum stress, protein folding and degradation, and the regulation of immune cell functions ( 42 – 44 ). In this study, XBP1 was downregulated in AS patients and negatively linked to neutrophil infiltration, suggesting that it may inhibit excessive neutrophil response under specific conditions, thereby alleviating inflammation. Section title: Discussion Educational score: 4.604621887207031 Domain: biomedical Document type: Study Language: en AS patients often exhibit iron metabolism disorders, such as anemia and iron overload, accompanied by enhanced oxidative stress and elevated lipid peroxidation levels, which are hallmark features of ferroptosis ( 16 , 45 ). The reduction in antioxidants, including glutathione (GSH) and antioxidant vitamins, observed in patients with AS mirrors the inactivation of the glutathione-dependent enzyme system in ferroptosis ( 46 ). This study found that the upregulation of the iron metabolism-related gene SLC40A1 and the lipid metabolism-related gene ACSL1 suggests that ferroptosis may play a critical role in the pathogenesis of AS. The upregulation of SLC40A1 may lead to intracellular iron accumulation, further exacerbating lipid peroxidation and activating inflammatory signaling pathways ( 37 ). Similarly, ACSL1 promotes ferroptosis by enhancing lipid oxidation, which significantly correlates with inflammatory markers such as hsCRP identified in this study ( 35 , 47 ). Additionally, the downregulation of antioxidant-related genes GZMM and XBP1 indicates decreased antioxidant capacity in AS patients, accelerating lipid peroxide accumulation and driving ferroptosis ( 44 ).In summary, this study reveals that ferroptosis may contribute to the pathology of AS through disruptions in iron metabolism, enhanced lipid peroxidation, and impaired antioxidant systems. Section title: Discussion Educational score: 4.344483375549316 Domain: biomedical Document type: Study Language: en Additionally, AS is not solely a chronic inflammatory disorder, but also displays features associated with autoimmune pathology ( 2 ). Research has indicated that neutrophils would accumulate at inflammation sites in AS, releasing various cytokines and chemokines that drive inflammation progression ( 48 – 50 ). Additionally, the proportion of activated CD8 T cells and dendritic cells is reduced in AS patients ( 7 , 51 ). This study analyzed the infiltration of 28 immune cell subsets in the AS group and the control group, identifying notable discrepancies in immune cell infiltration levels between the two groups, thereby reinforcing the autoimmune attributes of AS. We then examined the relationship between key genes and six significantly different immune cells, as well as the correlation between key genes and peripheral blood neutrophils. The results showed that the increase in neutrophils in AS was positively correlated with these genes. The study also found a decrease in the proportions of activated CD8 T cells, dendritic cells, TH1 cells, and γδT cells in AS patients, indicating that these cells are essential to the immune surveillance function in AS. Additionally, the AS group showed a notable increase in central memory CD8 T cells, which may reflect a sustained immune response and the establishment of memory cells. These findings provide new clues for further understanding the pathological mechanisms of AS. Section title: Discussion Educational score: 4.167407035827637 Domain: biomedical Document type: Study Language: en The key genes identified in this study (ACSL1, SLC40A1, GZMM, TRIB1, and XBP1) demonstrated favorable diagnostic performance and were associated with disease activity in AS patients. These biomarkers hold considerable potential for clinical applications. For instance, their expression levels could serve as sensitive indicators for early diagnosis, facilitating the identification of high-risk individuals prior to symptom onset. Additionally, dynamic monitoring of these genes’ expression levels may help evaluate disease activity and therapeutic responses, providing valuable guidance for personalized treatment strategies. Notably, genes associated with ferroptosis and immune infiltration, such as ACSL1 and SLC40A1, may serve as promising targets for future therapeutic interventions. Future research should focus on validating these biomarkers in larger, multi-center cohorts and standardizing their use in clinical practice to develop effective diagnostic tools or therapeutic approaches. Furthermore, these biomarkers may achieve greater predictive performance when combined with other clinical parameters, such as imaging or traditional inflammatory markers, thereby improving the accuracy of AS diagnosis and comprehensive disease management. Section title: Discussion Educational score: 4.043047904968262 Domain: biomedical Document type: Study Language: en Although this study provides new insights into the molecular mechanisms of AS, it has several limitations. First, the relatively small sample size may limit the generalizability of the results. Future research should address this limitation by replicating these results in larger, multi-center. Second, this study primarily relies on gene expression data from PBMCs, which may not fully capture pathological changes in other relevant tissues or cell types. Moreover, while gene functions were inferred through bioinformatics methods, experimental validation is required further to enhance the reliability and applicability of the study results. Section title: Conclusion Educational score: 4.08681583404541 Domain: biomedical Document type: Study Language: en The core genes identified in our study demonstrated high diagnostic performance in distinguishing AS patients from healthy individuals, and their expression levels were associated with PBMCs and disease activity. Additionally, this study disclosed a potential association between AS and ferroptosis. Also, multiple core genes and key pathways related to AS pathogenesis were identified through comprehensive analysis. These core genes may serve as potential biomarkers and targets for the diagnosis and treatment of AS. The findings of our study offered new insights that will enable a better understanding of the molecular mechanisms underlying AS and the development of innovative diagnostic and therapeutic strategies. | Review | biomedical | en | 0.999999 |
PMC11695278 | Section title: Introduction Educational score: 4.020230293273926 Domain: biomedical Document type: Study Language: en Intraoral radiography, utilizing the bisecting technique, is a fundamental diagnostic approach in dentistry with its precession relying significantly on accurate film positioning and the careful angulation of x-ray beam ( 1 – 3 ). A dental radiologist primary responsibility during this procedure is to ensure optimum film placement through meticulous assessment of both the horizontal and vertical angulation of the x-ray beam ( 2 ). Errors in angulation may lead to image distortions such as elongation or foreshortening of images ( 2 – 4 ). These technical inaccuracies not only reduce the diagnostic quality of radiographic images but can also result in misdiagnosis and treatment failures, while potentially increasing radiation exposure to patients ( 1 – 4 ). To address these issues radiographic landmarks are utilized to improve positioning accuracy and minimize the need for repeated exposure ( 2 ). Section title: Introduction Educational score: 3.703108549118042 Domain: biomedical Document type: Other Language: en Standard reference points for intraoral radiography are well-established, with the Ala-tragus line being a crucial guide for directing the x-ray beam in both maxillary and inferior border of mandible for mandibular radiographic exposures. Additionally various anatomical landmarks in the maxilla and mandible serve as standard reference points for x-ray beam alignment particularly in the bisecting technique ( 1 – 4 ) . Section title: Introduction Educational score: 3.942397117614746 Domain: biomedical Document type: Study Language: en However the event effectiveness of these landmarks may be limited by anatomical variations and overlapping structures. Studies have shown that using the ala-tragus line as an anatomical reference line in periapical radiographic projections ( 2 ) can significantly improve accuracy in periapical imaging compared to those where it was not utilized as a reference. Nonetheless, visualizing the imaginary ala-tragus line presented certain challenges: Clinicians may occasionally rely on the inferior or superior border of the tragus, experience confusion over the posterior reference points, and required substantial skill and training to orient the x-ray beam accurately to the area of interest. Frankfort's horizontal plane could also be used for maxillary teeth radiographic positions. Furthermore, barring the usage of inferior border of mandible, as a guidance plane, there are no specific radiographic reference lines available for mandibular teeth. Section title: Introduction Educational score: 3.834502935409546 Domain: biomedical Document type: Study Language: en In response to these limitations, a pilot study was conducted to evaluate the effectiveness of modified radiographic reference guidance termed “Nallan's Lines.” This study aimd to offer a more precise and reliable approach for image acquisition in the bisecting the angle technique. It compared the usage of the standard reference guides and the “Nallan Lines” for maxillary and mandibular intraoral radiographic projections. Section title: Concept of Nallan's lines Educational score: 3.7852208614349365 Domain: biomedical Document type: Other Language: en Nallan's lines or Modified radiographic reference lines are confined by specific anatomical boundaries within the mandible and maxilla. In the maxilla, the reference line is an imaginary horizontal line bisecting the triangular region defined by the Frankfort horizontal line, the superior border of the tragus of the ear and the ala of the nose. In the mandible, the line is a horizontal line drawn from there soft tissue of Point B to the gonion on the same side as identify it in this cephalometric analysis. It further bisects the line drawn from corner of mouth to angle of the mandible superiorly and inferior border of the mandible inferiorly . Section title: Study design Educational score: 4.148630619049072 Domain: biomedical Document type: Study Language: en A single-centre, prospective, cross-over study was conducted over a 5 month period a t the Radiology Lab of RAKCODS after obtaining necessary ethical approval. The study was designed as an in vitro preclinical investigation which involved 9 participants, including general dental practitioners and dental nurses who routinely make radiographic projections in the dental clinics independently. Undergraduate dental students were trained for dental radiology projections in the radiology lab only from the third year of dental course based on the course curriculum. This necessitated more training before they could make intraoral radiographic projections unassisted. Thus they were excluded from participation in the study. Each participant performed two rounds of radiographic procedures within a 30-day period. This allowed for proper training of the personal and no overlap of both tt old and the new projection techniques. Each participant made at least 10 radiographic projections using the bisecting angle technique on the phantom model with and without a LASER assisted gyroscopic device. The projections included selected maxillary and mandibular teeth within the anterior and posteriors. A phantom head equipped with upper and lower jaws was utilized to simulate the human oral cavity for the radiographic procedures. This provided a consistent and reproducible model for all participants, ensuring standardization across the study. Utilizing the Raosoft sample size calculator, the calculated sample size with a 5% margin of error and 95% confidence level is 90. Section title: Study design Educational score: 4.006613731384277 Domain: biomedical Document type: Study Language: en The participants were divided into two groups: Group A (conventional standard projections) and Group B (Nallan Lines). Group A performed intraoral digital radiography using a CCD sensor and Nallan DirectRay device, following the standard radiographic reference points like the ala-tragus line and the inferior border of mandible. Subsequently the same participants assigned as Group B were trained to use Nallan's Lines as radiographic reference and repeated the procedure. The results of both groups were compared to radiographic procedures performed by a specialist oral radiologist using gyroscope-enabled devices with and without usage of the Nallan's lines. The horizontal and vertical angulations for each of the teeth were followed as standard angulations for both the technique without any change. Section title: Study design Educational score: 1.9259812831878662 Domain: biomedical Document type: Other Language: en Nallan-DirectRay is a gyroscope enabled laser device mounted on the position indicating device of the dental radiographic machine. This was innovatively developed device for precise and accurate radiographic reference points on the area of interest. The validity of the device was checked and is under patent evaluation . Section title: Study design Educational score: 2.2505643367767334 Domain: biomedical Document type: Study Language: en The following equipment was employed for the study: • Intraoral x-ray Machine: Used for generating the radiographic images. • CCD Sensor: A charge-coupled device sensor was used to capture the x-ray images. • Nallan DirectRay Device: Utilized to assist in the accurate positioning of the x-ray beam. • Computer: For storing and analyzing the captured radiographs. • Lead Aprons: Provided to all participants to ensure radiation protection. Section title: Study design Educational score: 2.759347438812256 Domain: biomedical Document type: Other Language: en The landmarks used as guidance for performing radiographic examinations of maxillary and mandibular teeth with standard and Nallan's Lines are provided in Table 1 . Section title: Study design Educational score: 3.8520498275756836 Domain: biomedical Document type: Study Language: en The radiographs taken by each participant were saved as digital images and were transferred to a computer. The images were organized and analyzed using Microsoft Excel. Parameters such as image quality, clarity of anatomical landmarks, and technical aspects such as the length and breadth of the teeth projected, foreshortening, elongation, apical cut, cone cut and horizontal overlap were systematically recorded, evaluated and compared between the two groups. Section title: Statistical analysis Educational score: 4.050506591796875 Domain: biomedical Document type: Study Language: en The data were analyzed to evaluate the consistency and accuracy of radiographs taken by both groups. Statistical analysis was performed using SPSS for Windows, Version 16.0 (Chicago, IL). A paired Student's t -test was used to compare the mean length and breadth of the tooth (in millimeter) in both the control and study groups before and after training. The McNemar test was employed to compare radiographic errors before and after radiographic training in both groups. level of significance was set at p < 0.05. Bonferroni Correction and Fischer exact test were also performed address issues with small sample size. Intra observer variability obtained for oral radiologist images was 0.8 kappa value. Section title: Discussion Educational score: 4.059849739074707 Domain: biomedical Document type: Study Language: en This pilot study aimed to evaluate effectiveness of modified radiographic reference lines, termed “Nallan's lines”, compared to conventional standard lines (ala-tragus lines, Frankfurt horizontal plane) in intraoral periapical radiography. The study findings indicated that the implementation of Nallan's lines significantly improved the accuracy of radiographic images, particularly by reducing elongation of images, apical cut and horizontal overlap errors. This study aligned with Al-Safi who suggested developing a new method in the bisecting angle technique to enhance radiographic outcomes rather than relying on the traditional approaches. However, any specific new method or approach for radiographic projections were not discussed ( 2 ). The Nallan's lines could represent an evolution in this approach, offering a practical solution for dental practitioners aiming to improve radiographic precision and reduce errors. Section title: Discussion Educational score: 4.0671820640563965 Domain: biomedical Document type: Study Language: en The findings demonstrated a statistically significant improvement in the mean length measurements of teeth following radiographic training with Nallan's lines ( p < 0.01). This indicates that the modified reference lines enhance image positioning accuracy, contributing to more precise diagnosis and treatment planning based on radiographic images. The absence of significant changes in the control group emphasizes the limitations of traditional reference points which might be more susceptible to variations in anatomy and overlapping structures. Section title: Discussion Educational score: 4.127463340759277 Domain: biomedical Document type: Study Language: en Our results also showed substantial reductions in specific radiographic errors in the study group post-training. Notably, elongation errors decreased from 26.7% to 9.0% ( p < 0.003), and apical cut errors dropped from 45.6% to 8.9% ( p < 0.001). Horizontal overlap errors also significantly reduced ( p < 0.001). These improvements underscore the potential of Nallan's Lines to minimize common technical errors, thereby enhancing image quality and reducing the need for repeated exposures, which ultimately supports better patient safety by lowering cumulative radiation doses. Section title: Discussion Educational score: 4.101881980895996 Domain: biomedical Document type: Review Language: en A systematic review highlighted that the reject rates and error sources in dento-maxillofacial radiography, focusing on intraoral, extra-oral, and CBCT imaging. It also projected that rejection rates were highest for intraoral images, especially periapical radiographs (16.38%), primarily due to positioning errors and patient discomfort. This resonated with our work on Nallan's Lines, as improved positioning methods could reduce these issue significantly ( 6 ). In the case of extraoral radiography, the reject rate averaged nearly about 4% for panoramic images and 6% for lateral cephalograms, with patient movement as a main error source. The study also noted a 2.77% rejection rate for CBCT images, where patient stability and head position significantly impacted image quality, suggesting that clearer anatomical references and stability tools, similar to the goals of Nallan's Lines, could reduce errors ( 6 ). The review also underscored the need for accurate reference points, adequate operator training, and ergonomic adjustments in positioning, which alignd with our findings on Nallan's Lines' effectiveness in enhancing radiographic accuracy and reducing error rates, such as elongation and apical cut ( 6 ). Section title: Discussion Educational score: 3.6002423763275146 Domain: biomedical Document type: Study Language: en A study investigated the proficiency of general dental practitioners in Saudi Arabia with intraoral radiographic techniques. It highlighted variations in technique availability and ease of use, suggesting that standardized training could enhance accuracy in radiographic projections and image. It underscored the importance of precise training in radiographic positioning for improved diagnostic outcomes and reduced errors ( 7 ). Section title: Discussion Educational score: 3.3800389766693115 Domain: biomedical Document type: Review Language: en A more recent review demonstrated that though paralleling technique of dental periapical radiography was considered a better approach for endodontic and periodontal treatment plans, the technique might be a disadvantageous for Asian population group because of space insufficiency in maxillary region. Hence a more practical approach with different radiographic reference points were used in this study which may be employed for any population group ( 8 ). Section title: Discussion Educational score: 4.010861396789551 Domain: biomedical Document type: Study Language: en The present study was in agreement with the another study describing the periapical radiographic errors related to apical cut and image contrast ( 9 , 10 ). Some studies also demonstrated the need for check list or usage of rectangular collimation for dental radiographic projections in order to reduce the errors during the procedures and images obtained. It may be noted that though the check list might not had positive significant correlation between check list use and error occurrence, these new recommendations and modifications may improve the quality of images obtained thereby reducing patient radiation exposure ( 11 , 12 ). These findings are consistent with another similar study emphasizing the importance of accurate reference points in the intraoral radiography. Section title: Discussion Educational score: 3.115895986557007 Domain: biomedical Document type: Study Language: en The present study used an instrument called Nallan's DirectRay. It is a gyroscope enabled laser device mounted on the position indicating device of the dental radiographic machine. The device helped in precise placement of the position indicating device of the x-ray machine on the phantom model with a digital display of vertical angulations. The validity of the device was checked and under patent evaluation. Section title: Discussion Educational score: 3.9719526767730713 Domain: biomedical Document type: Study Language: en Shetty et al. ( 4 ) discussed the future potential of laser-guided intraoral radiography for improving accuracy in radiographic procedures. Similarly, Chau et al. ( 13 ) and Zamani et al. ( 14 ) demonstrated that using laser-guided indicators significantly reduced technical errors in radiography ( 15 ). Our study supported these findings by demonstrating that Nallan's lines, used in conjunction with gyroscopic laser guided approach, could serve purpose of enhancing accuracy. Section title: Discussion Educational score: 4.112551689147949 Domain: biomedical Document type: Study Language: en When comparing the study subjects to the specialist radiologist, the study group exhibited a closer approximation of the gold standard in terms of radiographic error rates, post-training. Although not all improvements where statistically significant, the trend suggested a positive impact of the modified reference lines on the radiographic accuracy. Despite the lack of statistically significant differences, several aspects suggested that Nallan's lines offer practical value that may not be fully captured by statistical results alone. Clinically, even modest reductions in radiographic errors—such as foreshortening, elongation, and apex cut could enhance diagnostic clarity and patient outcomes. After implementing Nallan's lines, we observed directional improvements across parameters, with increased rates of error-free images in study subjects, approaching the accuracy achieved by the radiologist. In the clinical context, these incremental improvements could be meaningful, particularly for less-experienced practitioners who may benefit from additional guidance in positioning techniques. Section title: Discussion Educational score: 3.965944528579712 Domain: biomedical Document type: Study Language: en Furthermore, Nallan's lines show significant educational potential as a training aid. Visual references, like those provided by this device, could help students and early-career clinicians develop better spatial awareness and technical accuracy, reinforcing proper positioning habits. This is especially valuable in training environments, where proficiency in radiographic positioning can be challenging to master quickly. While these trends may not appear statistically significant in the current study, repeated use and familiarity with Nallan's lines could, over time, lead to more consistent and accurate positioning, ultimately reducing the likelihood of errors in clinical practice. Section title: Limitations and recommendations Educational score: 4.0497541427612305 Domain: biomedical Document type: Study Language: en Future research may yield stronger evidence of the device's efficacy, especially with larger sample sizes or longer study durations. A larger cohort could provide the statistical power needed to detect smaller, yet clinically meaningful, effects that a limited sample size might obscure. Similarly, a randomized control trial assessing the device's impact over time could provide a more comprehensive understanding of its benefits, particularly if it focuses on practical improvements in radiographic accuracy within routine clinical settings. Another potential benefit lies in reducing the need for repeat exposures, which enhances patient safety by limiting unnecessary radiation exposure. This aligns with a patient-centric approach to healthcare, where even minor reductions in radiographic errors have broader implications for patient safety and care quality. Section title: Conclusion Educational score: 3.9145848751068115 Domain: biomedical Document type: Review Language: en The introduction of Nallan's lines as modified radiographic reference points demonstrated a substantial improvement in the accuracy of intraoral periapical radiographs, with particular effectiveness in reducing elongation, apical cut, and horizontal overlap errors. This advancement suggested that Nallan's lines may offer a robust framework for improving radiographic precision, leading to more accurate diagnosis and enhanced treatment planning. | Study | biomedical | en | 0.999996 |
PMC11695280 | Section title: Introduction Educational score: 4.032631874084473 Domain: biomedical Document type: Review Language: en Hypertensive disorders of pregnancy (HDP) are a group of maternal disorders characterized by rising blood pressure during pregnancy, with systolic blood pressure > = 140 mm Hg and/or diastolic blood pressure > = 90 mm Hg ( 1 ). The global observed prevalence of HDP was 5.2–8.2% ( 2 ). The prevalence of HDP varies greatly by region. For example, the prevalence of HDP was 7.30% in China ( 3 ), 7.44% in North China, 6.09% in Northeast China, 5.50% in Northwest China, 4.63% in East China, 3.20% in Southwest China, 2.59% in South China, and 1.23% in Central China ( 4 ). The prevalence of HDP was 10.6% in the United States ( 5 ), 6.73% in France ( 6 ), 8.5% in Denmark ( 7 ), 6.07% in Ethiopia ( 8 ), 8% in Sub-Saharan Africa ( 9 ). Section title: Introduction Educational score: 4.24409294128418 Domain: biomedical Document type: Review Language: en HDP has many adverse outcomes for pregnant women and their fetuses. On the one hand, in the general population, hypertension is one of the leading causes of death due to heart disease and stroke ( 10 ). On the other hand, pregnant women are a special population, and if they suffer from HDP, it can be harmful not only to the pregnant woman herself but also to their fetuses. For example, Roberts et al. reported that women with HDP were five times more likely to have perinatal death compared to women without HDP ( 11 ); Say et al. reported that HDP was one of the leading causes of maternal and fetal deaths ( 12 – 14 ) and that it occurred mostly in developing countries ( 8 ). Garovic et al. reported that HDP was associated with adverse fetal outcomes, such as preterm delivery, small for gestational age, low birth weight, etc., ( 15 ). In addition, HDP may cause long-term adverse pregnancy outcomes. For example, it is well-accepted that the frequency of hypertension is significantly higher after HDP, and that hypertension develops faster than in pregnant women with normal blood pressure; The earlier onset of cardio-metabolic risk factors and cardiovascular disease events, as well as higher rates of accumulated chronic conditions and multi-morbidity, support the thesis of accelerated aging among women with a history of HDP ( 15 ); Boucheron et al. found that sustained HDP exposure was an additional risk factor for chronic hypertension ( 6 ); Mito et al. found that HDP was a strong risk factor for the development of hypertension only 5 years after delivery ( 16 ); Egawa et al. found HDP was associated with cardiovascular disease in middle- and older-aged Japanese women ( 17 ); Goldstein et al. reported that HDP was associated with future heart failure risk, including peripartum cardiomyopathy, pregnancy-associated heart failure with preserved ejection fraction, and new-onset heart failure later in life ( 18 ); Kanata et al. reported that HDP could have a significant clinical impacts not only on the mother’s but also on the offspring’s health ( 19 – 21 ). Section title: Introduction Educational score: 3.2528724670410156 Domain: biomedical Document type: Study Language: en Due to the high prevalence and adverse outcomes of HDP, research on HDP is very significant and deserves more attention. There have been some studies on HDP, such as epidemiology, prevention, diagnosis, and management ( 2 , 3 , 22 – 26 ). However, some research could be added to this field. First, to the best of our knowledge, there are fewer studies on HDP in Hunan Province, China. Hunan Province is located in south-central China and covers a population of about 65 million. Compared with eastern China, Hunan Province is relatively underdeveloped ( 27 ). As mentioned above, most HDP-related maternal and fetal deaths occurred in developing regions. Second, although there have been some studies on the prevalence of HDP in China, to the best of our knowledge, there are few studies on the relationship between HDP and adverse pregnancy outcomes and risk factors for HDP. More studies need to be included in China. Section title: Introduction Educational score: 3.655512571334839 Domain: biomedical Document type: Study Language: en Therefore, in this study, we aim to describe the epidemiology of HDP, explore the relationship between HDP and adverse pregnancy outcomes, and explore the risk factors for HDP using long-term, large-area, and large-sample surveillance data from Hunan Province, China, 2012–2022. Our research will contribute to this field. Section title: Data sources Educational score: 4.093764305114746 Domain: biomedical Document type: Study Language: en This study used data from the Maternal Near-Miss Surveillance System in Hunan Province, China, 2012–2022, run by the Hunan Provincial Health Commission, and involves 18 representative registered hospitals in Hunan Province. These 18 hospitals are well-distributed throughout the province and are well-represented. In each of the 18 hospitals sampled, data were collected for all pregnant or post-partum women admitted to obstetrics departments. Surveillance data of all pregnant and post-partum women, including HDP, socio-demographic characteristics, obstetric history, and adverse pregnancy outcomes, were collected using an especially designed data collection form. Detailed information about the data collection process has been reported elsewhere ( 28 ). This study’s information collection methods and indicator definitions were consistent with the WHO standards ( 29 , 30 ). In this study, the adverse pregnancy outcomes included maternal deaths, maternal near-miss, preterm birth, stillbirth and neonatal death, low birth weight, hemorrhage disorder, infections, and other common diseases (such as heart disease, embolism, liver disease, anaemia, diabetes mellitus, renal disease, and pulmonary disease). Section title: Definition and classification of HDP Educational score: 3.870798349380493 Domain: biomedical Document type: Study Language: en The definition and diagnosis of HDP complied with the 2021 International Society for the Study of Hypertension in Pregnancy classification, diagnosis, and management recommendations for international practice ( 1 ). According to previous studies, HDP is usually classified into four categories: (1) preeclampsia-eclampsia, (2) chronic hypertension, (3) chronic hypertension with superimposed preeclampsia, and (4) gestational hypertension ( 3 , 31 ). Section title: Ethics approval and consent to participate Educational score: 1.8895502090454102 Domain: biomedical Document type: Study Language: en The Hunan Provincial Health Commission routinely collected surveillance data, and the government has developed the “Maternal Near Miss Surveillance Working Manual” to collect those data. Therefore, there is no additional written informed consent. The Medical Ethics Committee of Hunan Provincial Maternal and Child Health Care Hospital approved the study . It is a retrospective study of medical records; all data were fully anonymized before we accessed them. Moreover, we de-identified the patient records before analysis. We confirmed that all operations were following relevant guidelines and regulations. Section title: Data quality control Educational score: 1.7565120458602905 Domain: biomedical Document type: Other Language: en The Hunan Provincial Health Commission formulated the “Maternal Near Miss Surveillance Working Manual” for surveillance. Data were collected and reported by experienced and trained doctors and nurses. To ensure data consistency and accuracy, all collectors must be trained and qualified before starting work. The Hunan Provincial Health Commission asks the technical guidance departments to conduct comprehensive quality control yearly to reduce surveillance data integrity and information error rates. Section title: Statistical analysis Educational score: 4.073831081390381 Domain: biomedical Document type: Study Language: en We calculated the prevalence of HDP and 95% confidence intervals (CI) by the log-binomial method ( 32 ). Chi-square trend tests ( χ 2 trend ) were used to determine trends in prevalence by year. Unadjusted odds ratios (uORs) were used to examine the association between HDP and adverse pregnancy outcomes and demographic characteristics. Multivariate logistic regression analysis (method: Forward, Wald, α = 0.05) and adjusted odds ratios (aORs) were used to identify risk factors for HDP. We used the presence or absence of HDP as the dependent variable, and the demographic characteristics with significant uOR were entered as independent variables in multivariate logistic regression analysis. Section title: Statistical analysis Educational score: 1.3451675176620483 Domain: biomedical Document type: Other Language: en Statistical analyses were performed using SPSS 18.0 (IBM Corp., NY, USA). Figures were drawn using GraphPad Prism 9.5 (GraphPad Software, MA, USA). Section title: Prevalence of HDP Educational score: 4.111907482147217 Domain: biomedical Document type: Study Language: en Our study included 780,359 pregnant women, and 38,397 women with HDP were identified, with a prevalence of 4.92% (95% CI 4.87–4.97). Preeclampsia-eclampsia, gestational hypertension, chronic hypertension, and chronic hypertension with superimposed preeclampsia accounted for 46.31% (17,782 cases), 41.38% (15,889 cases), 8.70% (3,340 cases), and 3.61% (1,386 cases) of all HDP, respectively, and the prevalence was 2.28% (95% CI 2.25–2.31), 2.04% (95% CI 2.00–2.07), 0.43% (95% CI 0.41–0.44), and 0.18% (95% CI 0.17–0.19), respectively. Section title: Prevalence of HDP Educational score: 4.027612209320068 Domain: biomedical Document type: Study Language: en From 2012 to 2022, the prevalence of HDP increased from 3.11 to 7.39%, showing an upward trend ; the prevalence of preeclampsia-eclampsia ( χ 2 trend = 97.37, p < 0.01), gestational hypertension , chronic hypertension , and chronic hypertension with superimposed preeclampsia ( χ 2 trend = 192.45, p < 0.01) also showed upward trends . Section title: Relationship between HDP and adverse pregnancy outcomes Educational score: 4.028742790222168 Domain: biomedical Document type: Study Language: en HDP was associated with the following adverse pregnancy outcomes: maternal deaths (uOR =4.05), maternal near-miss (uOR =6.37), preterm birth (uOR =2.51), stillbirth and neonatal death (uOR =1.45), low birthweight (uOR =4.37), abruptio placentae (uOR =4.45), uterine atony (uOR =1.49), retained placenta (uOR =1.54), puerperal infections (uOR =2.14), abdominal surgical site infections (uOR =2.50), urinary tract infections (uOR =1.60), upper respiratory tract infections (uOR =1.75), heart disease (uOR =2.76), embolism (uOR =2.66), liver disease (uOR =1.25), anemia (uOR =1.38), diabetes mellitus (uOR =2.35), renal disease (uOR =4.66), and pulmonary disease (uOR =4.70, p < 0.05) . Section title: Epidemiology of and multivariate logistic regression analysis for risk factors for HDP Educational score: 2.6646647453308105 Domain: biomedical Document type: Study Language: en Table 3 shows the epidemiology of HDP. Univariate analysis shows that all variables in Table 3 are associated with HDP. Therefore, all variables in Table 3 were used as independent variables in the multivariate logistic regression analysis. As a result, all variables in Table 3 entered the regression model. Section title: Epidemiology of and multivariate logistic regression analysis for risk factors for HDP Educational score: 4.063729286193848 Domain: biomedical Document type: Study Language: en Compared to maternal age 25–29 years, HDP were less common in <20 years (aOR =0.60, 95% CI 0.53–0.69) or 20–24 years (aOR =0.84, 95% CI 0.81–0.87) and more common in 30–34 years (aOR =1.54, 95% CI 1.50–1.58) or > =35 years (aOR =2.77, 95% CI 2.69–2.86). Compared to the gravidity = 1, HDP were less common in gravidity = 2 (aOR =0.95, 95% CI 0.92–0.98) and more common in gravidity > = 4 (aOR =1.10, 95% CI 1.05–1.14). Compared to parity = 0, HDP were less common in parity = 1 (aOR =0.56, 95% CI 0.54–0.58) or > =2 (aOR =0.59, 95% CI 0.56–0.62). Compared to prenatal examination = 8–10 times, HDP were less common in prenatal examination <5 times (aOR =0.67, 95% CI 0.65–0.70) or 5–7 times (aOR =0.86, 95%CI 0.84–0.88). HDP was more common in previous cesarean sections than vaginal delivery (aOR =1.27, 95% CI 1.24–1.31). Section title: Epidemiology of and multivariate logistic regression analysis for risk factors for HDP Educational score: 4.061741828918457 Domain: biomedical Document type: Study Language: en There were significant differences in the results of univariate analysis and multivariate logistic regression analysis for some factors. For example, gravidity = 3 was a protective factor for HDP in the univariate analysis (uOR =0.95, 95% CI 0.93–0.98), while not in the multivariate logistic regression analysis (aOR =1.01, 95% CI 0.97–1.05). Parity > = 2 was a protective factor for HDP in the multivariate logistic regression analysis (aOR =0.59, 95% CI 0.56–0.62), while not in the univariate analysis (uOR =0.99, 95% CI 0.95–1.03). Prenatal examination > = 11 times was a protective factor for HDP in the univariate analysis (uOR =0.95, 95%CI 0.92–0.99), while not in the multivariate logistic regression analysis (aOR =0.98, 95% CI 0.94–1.02) . Section title: Discussion Educational score: 2.8885581493377686 Domain: biomedical Document type: Study Language: en Overall, we described the epidemiology of HDP, explored the relationship between HDP and adverse pregnancy outcomes, and explored risk factors for HDP. Our study is the most recent systematic study of the relationship between adverse pregnancy outcomes, risk factors, and HDP in China. Our research makes some original contributions to the field. There are several relevant findings in this study. Section title: Discussion Educational score: 4.142480373382568 Domain: biomedical Document type: Study Language: en First, the prevalence of HDP in this study was relatively high, which led to a considerable number of pregnant women being affected by HDP. The global prevalence of HDP was 5.2–8.2%, and the average prevalence in China was 7.30% ( 2 , 3 ), which is consistent with the 2022 prevalence in this study. However, as mentioned in the Introduction, there are significant differences in the prevalence of HDP in different countries or regions. For example, Ye et al. reported that the prevalence of HDP was 7.44% in North China, 6.09% in Northeast China, 5.50% in Northwest China, 4.63% in East China, 3.20% in Southwest China, 2.59% in South China, and 1.23% in Central China ( 4 ). Hunan Province is located in south-central China, and the prevalence of HDP in this study was significantly higher than in Ye’s study. In addition, the prevalence of HDP in this study showed a significant upward trend from 3.11% in 2012 to 7.39% in 2022. It is inconsistent with the decreasing trend in most countries and regions ( 33 ). The above findings may be associated with several factors. First, differences in the prevalence of HDP in different countries may be primarily related to variants in race, economics, and medical conditions. For example, Ward et al. found genetic variants related to HDP in different races ( 34 , 35 ). Due to limitations in economic and medical conditions, some less developed countries have relatively low HDP diagnosis rates ( 36 – 38 ), and fewer studies have been conducted, or only in a few hospitals, in a small area, and in a long time ago, which may be unrepresentative and lead to biased results ( 4 , 39 – 42 ). Second, the differences in the prevalence of HDP in different regions of China may be primarily related to variants in lifestyle, economic, and medical conditions. For example, Lu et al. found regional disparities in prenatal care and socio-economic in China ( 43 , 44 ), which may affect the diagnosis and treatment of HDP. Unhealthy urban lifestyles (e.g., alcohol drinking and late-night eating) exist in Hunan, which may increase the risk of HDP ( 45 ). Third, the upward trend in the prevalence of HDP may indicate an increase in some risk factors and may also be partly related to higher diagnosis rates due to improved medical conditions. For example, Umesawa et al. reported that the risk factors for HDP included body mass index, anemia, smoking, alcohol intake, education, maternal age, primipara, previous experience of pregnant complications, gestational diabetes mellitus, preexisting disease (such as diabetes mellitus), urinary tract infection, family history, and genetic variants ( 2 ). In recent years, the prevalence of many risk factors has increased, such as overweight and obesity ( 46 ), advanced maternal age ( 47 , 48 ), and diabetes mellitus ( 49 , 50 ). Especially after the implementation of the “two-child policy” in 2015, many risk factors have increased significantly ( 51 , 52 ). It may be the main reason for the upward trend of HDP prevalence in this study. In addition, as mentioned above, with the development of medical conditions ( 27 ), the diagnosis rate of HDP is gradually increasing, which may also be one of the reasons for the upward trend of HDP prevalence. Section title: Discussion Educational score: 4.077737808227539 Domain: biomedical Document type: Study Language: en In this study, we also reported the prevalence of preeclampsia-eclampsia (2.28%), gestational hypertension (2.04%), chronic hypertension (0.43%), and chronic hypertension with superimposed preeclampsia (0.18%). Li et al. reported that the prevalence of preeclampsia-eclampsia, gestational hypertension, chronic hypertension, and chronic hypertension with superimposed preeclampsia were 4.50, 3.30, 0.60, and 0.60%, respectively (China) ( 3 ). Umesawa et al. reported the prevalence of gestational hypertension was 1.8–4.4% (global) ( 2 ). The American College of Obstetricians and Gynecologists reported that chronic hypertension is present in 0.9–1.5% of pregnant women ( 53 ), and preeclampsia complicates 2–8% of pregnancies globally ( 54 ). In this study, the prevalence of chronic hypertension and chronic hypertension with superimposed preeclampsia was relatively low. It may be partly associated with missed or misdiagnosed cases, and this phenomenon is more common in areas with poor medical conditions. To the best of our knowledge, some previous studies of HDP in China did not provide a detailed classification of subtypes, and some did not use internationally recognized classification methods. In this study, the internationally recognized classification of HDP was used for the first time in Hunan Province, allowing for a comparison of the prevalence in different regions. Section title: Discussion Educational score: 4.032721996307373 Domain: biomedical Document type: Study Language: en Second, we found that HDP was associated with many adverse pregnancy outcomes. Previous studies have shown that HDP was associated with many adverse pregnancy outcomes, such as maternal deaths ( 12 , 13 ), maternal near-miss ( 55 ), low birthweight and preterm birth ( 56 ), stillbirth, neonatal death ( 57 , 58 ), abruptio placentae ( 59 ), retained placenta ( 60 ), puerperal infections ( 61 ), urinary tract infections ( 62 ), liver disease ( 63 ), and pulmonary disease ( 64 ). However, some of the adverse pregnancy outcomes in this study had been rarely addressed in previous studies, such as uterine atony, abdominal surgical site infections, upper respiratory tract infections, embolism, anemia, and connective tissue disease. In addition, this study provided the prevalence and risk values (ORs) of HDP in different populations. It may be helpful in clinical counseling. This is an observational study, and we cannot determine whether there is a causal relationship between HDP and adverse pregnancy outcomes. HDP may contribute to some adverse pregnancy outcomes, while some adverse pregnancy outcomes may be responsible for HDP. In-depth studies are needed to explore the mechanisms. This study is helpful for future research. Section title: Discussion Educational score: 4.029139518737793 Domain: biomedical Document type: Study Language: en Third, we identified several risk factors for HDP, including advanced maternal age, high gravidity, primipara, and previous cesarean section, and low-frequency prenatal examination was the only protective factor for HDP. Some risk factors have been widely accepted, such as advanced maternal age, primipara, and cesarean section ( 2 , 65 ). However, to the best of our knowledge, previous studies rarely addressed some factors, such as high gravidity and low-frequency prenatal examination. We infer that the high gravidity may be associated with some pregnancy complications, such as recurrent miscarriages ( 66 ), which may be associated with HDP ( 67 , 68 ). The low-frequency prenatal examination may be mainly associated with poor economic and medical conditions and low education, which may cause a relatively low diagnosis rate of HDP. It has been discussed above. Similar to the relationship between HDP and adverse pregnancy outcomes mentioned above, the relationship between HDP and the above factors could not be determined to be causal. Section title: Discussion Educational score: 2.460681200027466 Domain: biomedical Document type: Other Language: en Overall, the prevalence of HDP was relatively high in Hunan Province, and we have identified several risk factors for HDP. It may contribute to public health interventions or prenatal care strategies. For example, to avoid HDP, we advise that women try to avoid getting pregnant at an advanced age. The government can implement public health programs for early diagnosis and free treatment of HDP among pregnant women with high gravidity or previous cesarean section, or primiparous women, to reduce the patients’ burden. Section title: Discussion Educational score: 2.397235631942749 Domain: biomedical Document type: Study Language: en This study could have been improved. First, due to data limitations, some factors, such as body mass index, lifestyle, economic conditions, education, family history, and race, were not included. Second, this was an observational study, and we could not determine whether there was a causal association between HDP and adverse pregnancy outcomes and demographic characteristics. Third, the prevalence of HDP may be slightly underestimated. This study is helpful for future research. Section title: Conclusion Educational score: 2.206089735031128 Domain: biomedical Document type: Study Language: en The prevalence of HDP was relatively high in Hunan Province. HDP was associated with many adverse pregnancy outcomes. Advanced maternal age, high gravidity, primipara, and previous cesarean section were risk factors for HDP. | Review | biomedical | en | 0.999996 |
PMC11695282 | Section title: 1 Introduction Educational score: 1.5829488039016724 Domain: other Document type: Other Language: en Social media are online community platforms and apps that let users create, share, and interact with each other's content. Social media content can be text, photos, videos, GIFs, audio, etc. Social media can be used for various reasons, from users who share interests communicating to getting informed about current worldwide events. Social media can also be used for detecting early signs of stress and depression . The existence of social media in our day-to-day lives is more prevalent than ever. As of 2023, there are roughly 4.9 billion social media users, a percentage that is more than 60% of the entire population and more than 100 social media platforms. X, previously known as Twitter, stands out as one of the most widely recognized social media platforms. Twitter was launched in 2006. It revolves around the concept of “following” other users. A user can follow accounts they are interested in and see their tweets in their timeline (“following”) and conversely can have “followers” that see their tweets. Nowadays, it has been established as a powerful tool for real-time news updates, public discourse, and social movements, and continues to evolve and enhance its user experience. In 2023, Twitter was renamed to X by then-CEO Elon Musk. The extensive presence of social media in the modern landscape has led to the emergence of accounts that automate interactions on social media platforms, often mimicking human behavior, the so-called bots. These bots can be coded to perform a variety of tasks, such as automatically publishing content, liking, sharing, following, or commenting on posts. Some can even be programmed to engage in conversations to promote specific agendas. Their behavior differs depending on their intent and purpose, but they might share features, such as very high or very low activity levels and more structured and characteristic language patterns . Uyheng et al. examined the origin and traits of trolling messages, finding that they often originate from automated bots and are distinguished by their use of abusive language, reduced cognitive complexity, and specific targeting of individuals or entities. Their study also noted a tendency for bots to target right-leaning sources of information, while trolls tended to engage with less polarized content, spreading misinformation across diverse audiences. Bots are very efficient in spreading misinformation, particularly when programmed with optimized values for factors like walking speed, network distribution, and strategy . Fake news and bots have had significant tangible consequences in several cases. Users tend to believe conspiracy theories and misinformation, and correction attempts can sometimes backfire . Users might also share fake news for altruistic or self-promotional purposes, yet those with greater social media literacy are better equipped to identify and refrain from spreading fake news . Therefore, there's a need for measures to promote truthful reporting in media and detect any cases of misinformation dissemination. Section title: 1 Introduction Educational score: 1.3662787675857544 Domain: other Document type: Study Language: en The need to detect bot accounts to shut them down is quite immediate, assessing the hazards of their uncontrollable presence on social media. Several studies to identify bots from real users have been conducted that provide satisfactory results. There have been several approaches, including supervised learning , unsupervised learning , reinforcement learning , and GNN-based architectures . However, all these traditional neural architectures often rely on fixed parameters that are manually designed. Constructing efficient neural network architectures requires extensive feature engineering and can be a quite challenging and time-consuming procedure. Also, fixed architectures often mitigate the models' adaptability on other datasets and tasks. Motivated by these limitations, we examine the implementation of Neural Architecture Search (NAS) to automate the process of discovering optimal architectures. NAS explores a search space of possible architectures and identifies the configurations that enhance the model's performance. Section title: 1 Introduction Educational score: 3.9876320362091064 Domain: biomedical Document type: Study Language: en A Neural Architecture Search method that has been proposed to solve the performance issues of fixed architectures is Deep and Flexible Graph Neural Architecture Search (DFG-NAS) . It employs an evolutionary algorithm to explore a vast space of permutations of Propagation and Transformation operations, to find the one with the best accuracy in the validation set. Addressing the limitations of previous bot detection models due to their fixed architectures we employ DFG-NAS on a GNN-based approach for bot detection. This approach leverages the user's semantical and property information and constructs a heterogeneous graph out of the follower-following relationships between users. Then, we adapt the DFG-NAS approach to handle Relational Graph Convolutional Neural Networks (RGCNs). The model automatically searches for the permutation of Propagation (P) and Transformation (T) functions, the two main processes of the message-passing protocol, with the highest validation accuracy. The model is also amplified with the use of the Gate operation on the P connections and the use of the skip-connection operation on the T connections. Section title: 1 Introduction Educational score: 1.121592402458191 Domain: other Document type: Other Language: en To the authors' knowledge, DFG-NAS has not been employed before in the task of bot detection. All our experiments were performed on the Twibot-20 dataset . The following sums up the contributions of our work: Section title: 2 Research objective Educational score: 1.1931897401809692 Domain: other Document type: Other Language: en It is evident to any social media user that bots continue to dominate the digital landscape despite extensive efforts in bot detection and platform initiatives to stop their activities. As technology advances, bots are programmed to mimic human mannerisms more effectively, making them more resilient against detection mechanisms. Beyond the irritation they pose to everyday users, some bots can have tangible and detrimental effects on human society. In 2016 fake news stories spread widely during the U.S. presidential election campaign aiming to influence the public vote . Throughout the COVID-19 pandemic , bots spread misinformation about the virus and the vaccines on social media, leading to mob panic, confusion, and even resistance to public health measures. Fake news is often framed in a manner that fosters negativity in social discussions and hinders individuals' ability to consider diverse perspectives, contributing to the formation of “echo chambers” on social media platforms . Bots also exacerbate cyberbullying by mass-targeting users, leading to serious psychological consequences. Social media platforms face challenges in effectively moderating such content. Cyberbullying detection methods often rely on unclear definitions and are prone to biases in data annotation . Their evolving nature raises concerns about the efficiency of current preventive measures, highlighting the need for innovation to prevent the dangers posed by this digital phenomenon. Section title: 2 Research objective Educational score: 2.1213467121124268 Domain: other Document type: Study Language: en The motivation for this research was constructing a model characterized by adaptability across future datasets, ensuring resilience in the face of evolving technology through time. Many contemporary models rely on fixed architectures, often struggling to demonstrate their efficiency on novel datasets. Although Neural Architecture Search (NAS) has shown significant advantages in various test cases, its application to bot detection remains relatively underexplored, with limited but promising results noted in studies such as Yang et al. . Considering the dynamic nature of the social media landscape and the continuous evolution of bots, more flexible architectures specifically designed for bot identification could offer a practical solution to mitigate their real-world consequences. Section title: 2 Research objective Educational score: 1.2353616952896118 Domain: other Document type: Other Language: en This research aims to showcase the efficiency advantages of architecture search and perhaps pave the way for more implementations of NAS models in bot detection in the ongoing battle against automated malicious activities. Section title: 3.1 Bot and fake news detection models Educational score: 1.8856892585754395 Domain: other Document type: Other Language: en The task of bot identification has attracted numerous studies and many state-of-the-art models propose fascinating methodologies. We could mainly divide these models into supervised learning approaches, unsupervised learning approaches, and GNN-based approaches. In this section, we present some baseline models proposed for bot detection and discuss how they fall into the above categories. Section title: 3.1 Bot and fake news detection models Educational score: 3.8314435482025146 Domain: other Document type: Review Language: en Lee et al. applied various machine learning algorithms, including SVMs, Naive Bayes, and decision trees, to build and evaluate a supervised bot detection model. The features used in their analysis included account-based features (e.g., the number of followers, friends, tweets), temporal features (e.g., time of account creation, tweet frequency), and content-based features (e.g., usage of URLs, hashtags). Kudugunta and Ferrara suggested a deep learning model that uses the user's tweets and some metadata features. This architecture includes a tokenizer, GloVE embedding layer, LSTM, and Dense layers. Wei and Nguyen used only users' tweets with no context of prior knowledge on user profiles, friendship networks, or behavior. They proposed a recurrent neural network (RNN) model that used word embeddings to encode tweets, a three-layer Bidirectional LSTM (BiLSTM), and a softmax layer at the binary output. Cai et al. proposed their model (BeDM) that involved deep neural networks in bot detection. They employed convolutional neural networks (CNNs) and LSTM, using only the tweet semantics, such as the frequency and the type of publications. Botometer is a web-based program developed by Davis et al. at Indiana University. It leverages more than 1,000 features to classify Twitter accounts as bots and humans, such as friends, the structure of the social network, user meta-data, temporal activity, and sentiment analysis. Botometer distinguishes the accounts by an overall bot score (ranging from 0 to 5), along with several other scores. The greater the score, the greater the probability that this account is linked to a bot. Yang et al. presented a thorough introduction of the latest version of Botometer for new users and demonstrated a case study. Alarfaj et al. utilized features based on content attained from the Twitter API and employed state-of-the-art classifiers, like MLPs, random forest, and naive Bayes. Features included messages, special characters, sentiment analysis, etc. Alothali et al. introduced their framework, called Bot-MGAT, which stands for bot multi-view graph attention network. The scientists pointed out that other approaches couldn't adjust to different datasets since there wasn't enough recently updated labeled data, which made sense given the constantly shifting behavior of the bots. They presented a methodology that makes use of transfer learning (TL) to leverage the multi-view graph attention mechanism. The framework also benefited from semi-supervised learning, using labeled and unlabeled data. The authors used the TwiBot-20 due to its graph structure, extracting 18 features for the training. Feng et al. suggested SATAR. In particular, SATAR leverages the user's semantics, property, and neighborhood information. It adjusts by fine-tuning parameters and pre-training on a huge number of self-supervised users. The authors proposed two alternative models: SATAR FC and SATAR FT . Ilias and Roussaki proposed two methods for bot detection using deep learning techniques. Their first approach extracts a substantial 71 features per user to utilize for account classification to bots and genuine users. They also employed various feature selection techniques to discard redundant and irrelevant features. Their second methodology proposes a deep learning architecture for tweet-level classification. This architecture incorporates an attention mechanism atop the Bidirectional Long Short-Term Memory (BiLSTM) layer. During the learning phase, the attention mechanism helps the model better focus on relevant information. Ilias et al. focused solely on user descriptions and sequences of actions performed by Twitter accounts. Their approach includes both unimodal (text or image) and multimodal (both text and image) methods. They designed digital DNA sequences per user based on tweet type and content, converted these sequences into 3D images, and fine-tuned pre-trained vision models like AlexNet, ResNet, and VGG16. For bot detection through user descriptions, they fine-tuned TwHIN-BERT, a transformer model. In multimodal approaches, they use VGG16 for visual representation and TwHIN-BERT for textual representation, proposing three fusion methods: concatenation, gated multimodal unit (GMU), and cross-attention. They conducted their experiments on both the Cresci'17 and TwiBot-20 dataset. Wei and Nguyen proposed their model BOTLE. Their model utilizes a recurrent neural network (RNN) with Bidirectional Gated Recurrent Units (BiLGRU) connecting two hidden layers of opposite directions leading to the same output. Notably, BOTLE does not rely on handcrafted features or pre-existing information regarding account profiles. Linguistic embeddings, including word, character, part-of-speech, and named-entity embeddings, are employed to encode tweet content, eliminating the need for labor-intensive feature engineering. Bazmi et al. introduced the Multi-View Co-Attention Network (MVCAN), which aims to capture the latent topic-specific credibility of both users and news sources. This model represents news articles, users, and news sources in a manner that encodes topical viewpoints, socio-cognitive biases, and partisan biases as vectors. These features are encoded using a variant of the Multi-Head Co-Attention (MHCA) mechanism. Shevtsov et al. introduced their model BotArtist, constructed on a semi-automatic machine learning pipeline, that requires minimal features for training, taking into consideration the loads of data needed by previous approaches and the recent monetization of Twitter API requests. Sujith et al. proposed a supervised learning approach that used multiple models to detect bots. Their classification of accounts relied on features like user metadata, tweet content, and posting history, among others. In addition to identifying bot accounts, the authors assigned a level of significance or influence to them, prioritizing the removal of the most influential or harmful bot accounts. Liu et al. proposed BotMoE, which leverages three perspectives of user information (metadata, text, and graph representations) and incorporates a community-aware Mixture-of-Experts (MoE) layer to assign users to different communities. The user representations are fused with an extractor fusion layer and supervised learning is employed to train the BotMoE framework to perform community-aware bot detection. Saxena et al. proposed two frameworks for recognizing accounts that disseminate false information on Twitter. Initially, they employed profile-based data, including the verified status, profile photo, and account lifetime and activity. Then, they combined tweet-propagation patterns and assigned a credibility score to each user, signifying their authenticity. Dimitriadis et al. proposed CALEB that is based on the Conditional Generative Adversarial Network (CGAN) and its extension, Auxiliary Classifier GAN (AC-GAN). By developing realistic artificial bot varieties, they were able to replicate the evolution of bots. As a result, they enhanced already-existing datasets and were able to identify bots before they emerged. Section title: 3.1 Bot and fake news detection models Educational score: 4.044909477233887 Domain: biomedical Document type: Review Language: en Yang et al. used a combination of unsupervised and supervised learning methods for bot detection. Specifically, the authors utilized minimal features derived from user metadata, temporal patterns, network structure, sentiment analysis, and linguistic cues that they fed into a machine learning pipeline, that reduced dimensionality and included classification algorithms. Cresci et al. introduced the Social Fingerprinting technique for bot detection, a Digital DNA technique that models social network users' behaviors. Each user is represented as a sequence of characters depending on the type and content of the tweets they publish, simulating a DNA sequence. The authors try to find similarities in the sequences defining the length of the Longest Common Substring (LCS) between two sequences. For a set of real users, the length of LCS was found to be particularly small, leading to the conclusion that longer sequences than the average LCS were bots. Based on this idea, the authors developed two techniques, one based on supervised learning and another on unsupervised learning to find similarities in the behavior of accounts. Quezada et al. developed a real-time bot infection detection model that analyzes Domain Name System (DNS) traffic events. They extracted 13 attributes from DNS logs to create unique fingerprints for servers. Using Isolation Forest, an algorithm for unsupervised learning, they identified anomalies in the fingerprints to classify hosts as infected or not. The model also utilized Domain Generation Algorithms (DGA) to search for queries to anomalous domains. Finally, a Random Forest, a supervised learning algorithm, was employed to create a model for detecting future bot infections on hosts. Miller et al. approached bot identification as an anomaly detection problem. They extracted 107 features from user's tweets and property information and adapted two stream clustering algorithms, StreamKM++ and DenStream, to facilitate spam detection and identified bot users as abnormal outliers. Chavoshi et al. developed DeBot, a bot detection system for social media, using warped correlation to identify likely bot accounts based on their high synchronicity, a characteristic unlikely in human users. DeBot doesn't require labeled data and operates on activity correlation. Moreover, through the utilization of a lag-sensitive hashing technique, it can promptly cluster accounts for real-time classification. Minnich et al. proposed their real-time unsupervised model BotWalk. Using metadata, content, temporal, and network features they employ anomaly detection, comparing each user to a seed bank of labeled accounts iteratively. Mannocci et al. proposed MulBot, an unsupervised bot detection system that utilizes multivariate time series (MTS) analysis. They employed an LSTM autoencoder to map the MTS data into a latent space and then conducted clustering on this encoded data to find dense clusters of users exhibiting similar behavior, assuming this was a common trait of bot accounts. MulBot also showcases effectiveness in identifying and distinguishing various botnets. Wu et al. employed unsupervised machine learning techniques, specifically K-Means and Agglomerative clustering, for Twitter bot detection. They used account activity, popularity, and verification status, among other features for the clustering. Koggalahewa et al. introduced an unsupervised method for bot identification based on a user's peer approval in the social network. They based peer acceptance between two users on their shared interests over a multitude of issues. Lopes et al. introduced their botnet identification model, designed to detect networks of compromised devices under master control. Their approach relies on analyzing network flow behavior through a contemporary method known as the Energy-based Flow Classifier (EFC). EFC employs inverse statistics to enhance anomaly detection. Section title: 3.1 Bot and fake news detection models Educational score: 3.5382988452911377 Domain: other Document type: Study Language: en Ali Alhosseini et al. introduced the use of graph convolutional neural networks (GCNN) in bot identification. They noted that besides the users' features, the construction of a social network would enhance a model's ability to distinguish the bots from the genuine users. Feng et al. introduced the aspect of diversity in relationships and influence dynamics among users in the Twittersphere for bot detection. They proposed a bot detection framework that leverages a network with users as nodes and the different relations as edges. Then they aggregated messages across users and operated heterogeneity-aware Twitter bot detection. They conducted their experiments using the Twi-Bot20 dataset. Feng et al. proposed their model for bot detection BotRGCN, which is short for Bot detection with Relational Graph Convolutional Networks. BotRGCN builds a heterogeneous graph out of the following relationships and uses information, such as the user's description, tweets, numerical and categorical property set, and neighborhood information. The experiments were conducted on the Twi-Bot20 dataset , but BotRGCN could exploit other types of relations if supported by the dataset. Kušen and Strembeck examined the structural dynamics of conversations between humans and bots on Twitter following emotionally charged riot events. They introduced “emotion-exchange motifs” to identify recurring patterns in emotional message exchanges. Their findings revealed that human conversations exhibited various motifs with reciprocal edges and self-loops, indicating interactive dialogue. In contrast, bots typically disseminated identical messages to multiple users or did not anticipate replies. Moreover, bots frequently initiated conversations and often conveyed fear-inducing messages. Bui and Potika introduced a graph-based method for bot detection. They detailed their data collection process and identified specific behaviors indicative of an account being associated with a bot. These behaviors can include engagement with other users, nonsensical usernames and profile information, repetitive content posting, and retweeting activity. These observations are utilized to label the accounts accordingly. Dehghan et al. suggested that the local social network formed around each account can aid in identifying the bots. To prove their hypothesis, they compared two classes of embedding algorithms, the former of which focused on proximity data and the latter that focused on nodes' neighborhoods. They discovered that the structural embeddings presented higher information underlining the valuable information that is embedded within each node's local network. Pham et al. introduced their approach Bot2Vec, which eliminated the need for user profile features. To improve the model's generalization on many social media platforms, they used only local neighborhood relations and the community structure of the graph that represented the users and employed an random walk strategy in the communities. Noekhah et al. proposed their model “Multi-iterative Graph-based opinion Spam Detection” (MGSD) that aims to identify various types of spam entities. It analyzes all kinds of relationships between them and utilizes domain-independent features, allowing for generalization across types of opinionated documents. Trained on both existing and novel features, MGSD assigns a spam score to each entity. Ye et al. proposed HOFA, a graph-based framework for bot detection, featuring two key modules: Homophily-Oriented Graph Augmentation (Homo-Aug) and Frequency Adaptive Attention (FaAt). The Homo-Aug employs an MLP to extract user representations and generate a k-NN graph. Meanwhile, the FaAt module acts as a low-pass filter for homophilic edges and a high-pass filter for heterophilic edges. This function prevents excessive smoothing of user features by the neighborhood. El-Mawass et al. explored using the output of existing supervised classification systems to detect spammers. They incorporated the classifiers' outputs as prior beliefs within a probabilistic graphical model framework. Proposing a bipartite users-content interaction graph, they facilitated the spread of beliefs to similar accounts. Constructing a Markov Random Field on a graph of similar users, they employed Loopy Belief Propagation to derive the predictions. Their findings demonstrated a notable enhancement in recall while maintaining precision. Section title: 3.2 Neural architecture search approaches Educational score: 1.6617063283920288 Domain: other Document type: Other Language: en Neural architecture search can increase performance in many tasks . Graph neural architecture search is proposed as the solution to performance limitations due to a fixed architecture. Parameter tuning in neural networks can be a challenging task. Many NAS methods have been suggested that include variations in the search space, the optimization method, and the architecture evaluation. We will divide these methods based on their optimization method, which will include reinforcement learning, evolutionary algorithms and gradient-based methods. Section title: 3.2 Neural architecture search approaches Educational score: 4.179618835449219 Domain: biomedical Document type: Study Language: en Zhou et al. proposed the automated graph neural networks (Auto-GNN) framework. Auto-GNN searches for the best GNN architecture possible in a predetermined search space, divided into six classes of actions: hidden dimension, attention function, attention head, aggregate function, combine function, and activation function. For efficiency reasons, the authors designed a conservative explorer to preserve the optimal neural architecture discovered during the search. The authors also implemented constrained parameter sharing, adapted to the heterogeneous GNN architecture. Two experimental methods were presented: inductive, in which the graph structure and node features on the testing and validation sets are unknown during training, and transductive, which involves the availability of unlabeled data for testing and validation during training. Gao et al. proposed GraphNAS to implement an automatic search of the best graph neural architecture based on reinforcement learning. The search space covers sampling functions, aggregation functions, and gated functions. GraphNas also uses more efficient parameter-sharing techniques than other contiguous models for CNNs and RNNs. After training 1,000 different architectures, the five best ones were used for the testing, which surpassed human-invented ones or those produced by random searches. Zhao et al. proposed the SNAG framework (Simplified Neural Architecture Search for Graph neural networks). The suggested framework had two key components: Node aggregators, which focused on neighborhood features, and Layer aggregators, which focused on the range of the neighborhood used. The search space algorithm was a variant of Reinforcement Learning that adopted the weight-sharing mechanism (SNAGWS). Nunes and Pappa presented one NAS methods for optimizing GNNs based on reinforcement learning and one based on evolutionary algorithms. The authors defined two cases of search spaces: Macro, where the architectures generated have the same structure, and Micro, where the architectures are not rigidly structured but combine several convolutional schemas. They concluded that EA and RL found very similar architectures to those found by a random search, a significantly simpler technique. However, they pointed out that whilst the other approaches generated large structures in as much as 80% of the situations, EA created the majority of GPU-fitting designs. Li et al. proposed Meta-GNAS that uses meta-reinforcement learning from past tasks to apply that knowledge to new tasks. Additionally, they speed up the search by using a predictive model to evaluate the potential graph neural architectures instead of training them from scratch. Section title: 3.2 Neural architecture search approaches Educational score: 4.448890686035156 Domain: biomedical Document type: Review Language: en Peng et al. implemented a NAS approach to human action recognition from skeleton movements. The search space was enlarged with diverse spatial-temporal graph modules while constructing higher-order connections between nodes using Chebyshev polynomial approximation. The search algorithm used is an evolutionary adaptation with a high sampling efficiency, denoted Cross-Entropy method with ImportanceMixing (CEIM). Jiang and Balaprakash adapted the method of neural architecture search to the conception of GNNs for predicting molecular properties. The authors designed neural networks for message-passing (MPNNs) between nodes. To find an optimal MPNN from the user-defined search space, they used regularized evolution (RE) from the DeepHyper package. Zhang et al. proposed DFG-NAS, an innovative method that allows for automatic search of very deep and adaptable GNN architectures. DFG-NAS focuses on exploring macro-architectures, specifically the implementation details of atomic propagation (P) and transformation (T) operations within the GNN. P is linked to the graph structure, whereas T concentrates on the non-linear transformations within the neural network. In addition, they adopted gating and skip-connection mechanisms for deeper GNN pipelines. They used an evolutionary algorithm to find the optimal architecture, which supported four cases of mutation. Peng et al. introduced Fast-ENAS as a computationally efficient alternative to Evolutionary Neural Architecture Search. This method utilizes a training-free performance metric that is computed with a single forward pass. The authors enhance the search process by incorporating a GCN-based contrastive predictor, aiming to improve the accuracy of the estimated performance of a candidate architecture, bringing it closer to its actual performance. Shang et al. introduced AG-ENAS, which brings two key innovations to the Evolutionary Neural Architecture Search process. Firstly, it employs an adaptive parameter adjustment mechanism based on population diversity and fitness, enhancing the adaptation of genetic operators' associated parameters. Secondly, the model introduces a mutation operator guided by the gene potential contribution. It improves offspring quality by assigning weight to more valuable genes through a distribution index matrix. The concept of aging is integrated into environmental selection to mitigate premature convergence. Lopes et al. presented Guided Evolutionary Architecture (GEA), which tackles the problem of other NAS models getting trapped in suboptimal solutions during the search process. GEA overcomes this challenge by generating and evaluating multiple architectures using a zero-proxy estimator and selecting only one with the best-performing one for the next generation. This approach expands the search space without increasing complexity, as new architectures are derived from previous ones through mutations. Section title: 3.2 Neural architecture search approaches Educational score: 4.060886859893799 Domain: biomedical Document type: Study Language: en Zhao H. et al. proposed their framework SANE. The search space has similarities with the search space from the SNAG framework, with Node and Layer aggregators. However, the authors presented a novel differentiable search algorithm. Cai et al. introduced a GNAS approach featuring a uniquely designed search space and a gradient-based search approach. The authors developed a three-level Graph Neural Architecture Paradigm (GAP) that includes two types of fine-grained atomic operations (neighbor aggregation and feature filtering) that are derived from message-passing, to build the search space. Li et al. introduced an innovative dynamic one-shot search space designed for multi-branch neural architectures within GNNs. The dynamic nature of the search space offers a larger capacity than a larger predefined search space. The architectures with lower importance weights are removed periodically from the population, while the candidate operations are unique to every edge of the computational graph. The authors performed both supervised and unsupervised techniques for the training part. Zhao J. et al. proposed a gradient-based architecture search method for predicting a system's remaining useful life. Their approach models the search space as a directed acyclic graph (DAG), where nodes represent latent representations and edges represent transformation operations. By employing candidate operations like ReLU and tanh, along with the softmax function, they make the search space continuous and the objective function differentiable, facilitating gradient-based optimization methods to find the optimal architecture. Section title: 3.3 Related work review findings Educational score: 1.5717138051986694 Domain: other Document type: Other Language: en From the aforementioned research works, it is clear that there have been many approaches to the task of bot detection. Previous studies include supervised, unsupervised, and graph neural network (GNN) based methods. While they have shown promising results, the relentless evolution of bot accounts toward simulating human-like patterns poses a significant challenge to their effectiveness. These models are constrained by fixed architectures, limiting their adaptability to newer datasets. Section title: 3.3 Related work review findings Educational score: 2.877126455307007 Domain: other Document type: Study Language: en Little work has been done in employing Neural Architecture Search methods in GNN-based methodologies for bot detection. Our work shifts the focus on overcoming the performance limitations due to fixed architectures, by utilizing DFG-NAS to search for the best configuration of Propagation and Transformation functions in the message passing protocol of our RGCNs. Instead of extensive feature engineering our model searches for the permutation with the highest accuracy and aims for better adaptability in newer datasets that will depict future bots' behavior. Moreover, DFG-NAS presents high advantages, as it is suitable for GNN-based methods and overcomes over-smoothing and model degradation issues with the gate and skip-connection operations. Section title: 4 Dataset Educational score: 1.3935563564300537 Domain: other Document type: Other Language: en The TwiBot-20 Dataset is a publicly available dataset, constructed with a breadth-first search (BFS) methodology. The dataset includes information about each user's profile information obtained from the Twitter API, recent tweets, and domains of the user's interest. It also contains information about the user's neighborhood, which helps us construct a heterogeneous graph from the following relationships. Table 1 presents all the attributes of the TwiBot-20 Dataset and a short description of them. The information from the user profiles is further mentioned in the preprocessing part of the model. The graph that is constructed consists of 229,580 nodes and 227,979 edges. The objective of the bot detection system is to distinguish between bots and genuine users by analyzing information from user descriptions, tweets, numerical and categorical properties, as well as neighborhood information. Section title: 5 Methodology Educational score: 1.8142486810684204 Domain: other Document type: Study Language: en In this part, we present a complete analysis of our methodology. First, we describe the preprocessing of the user metadata used in our model. Next, we introduce the use of Relational Graph Convolutional Neural Networks and the two functions in Message Passing. Last, we explain the use of DFG-NAS in searching for the best permutation of Propagation and Transformation functions. In Figure 1 , we depict the architecture of the model on a higher level, while Figure 2 presents the connections between the different layers of an example configuration of P and T functions. Section title: 5.1 Data preprocessing Educational score: 1.42421293258667 Domain: other Document type: Other Language: en We follow the preprocessing suggested by Feng et al. for BotRGCN. Each user's representation includes metadata that are preprocessed as follows: Section title: 5.1 Data preprocessing Educational score: 1.0782653093338013 Domain: other Document type: Other Language: en where D is the user embedding dimension. Each feature's procession and representation are explained below. Later we will prove that the model's performance is attributed to all these features and not only to the heterogeneous graph. Section title: 5.1 Data preprocessing Educational score: 1.1422839164733887 Domain: other Document type: Other Language: en where b ¯ denotes the user description representation and D s is the dimension of the RoBERTa embedding. The vectors for the user's description are derived: Section title: 5.1 Data preprocessing Educational score: 1.7923005819320679 Domain: other Document type: Other Language: en where W B and b B represent trainable parameters, ϕ denotes the activation function, and D is the dimension of the embedding. Section title: 5.2 Relational graph convolutional neural networks Educational score: 1.263916015625 Domain: other Document type: Other Language: en Our method builds a heterogeneous graph out of the following relationships. Users are considered nodes and the “following” and “followers” relations are represented as edges connecting the nodes. The user's “followers” are therefore represented differently than the user's “following.” The heterogeneous graph that is constructed can represent better the relations between users and more relations between the users could be integrated into the graph if supported by the dataset. The users also contain the concatenated metadata that we described below. Section title: 5.2 Relational graph convolutional neural networks Educational score: 1.0775809288024902 Domain: other Document type: Other Language: en To combine the users' representations with the relationships between users we make use of RGCNs. The message-passing process in RGCNs comprises two fundamental operations: propagation (P) of the representations of the user's neighbors and transformation (T) on these representations. Below we describe the process behind the two functions: Section title: 5.2 Relational graph convolutional neural networks Educational score: 1.271799921989441 Domain: other Document type: Other Language: en We segregate these two types of functions since combinations of them will construct the search space for the architecture search. Section title: 5.3 Graph neural architecture search Educational score: 2.551520347595215 Domain: other Document type: Study Language: en The use of Graph Neural Networks offers undeniable advantages in the task of bot detection. However, maximizing their performance may require extensive feature engineering. This is why we employ Graph Neural Architecture Search, using the model DFG-NAS . Thus, we search for the permutation of Propagation and Transformation steps that achieves the highest accuracy. Most G-NAS methods have a fixed pipeline length since the performance decreases with too many P operations as the layers become deeper, which is referred to as the over-smoothing issue. Propagation and transformation operations regulate the effect of smoothing. Moreover, with unlimited pipeline length DFG-NAS searches for more flexible pipelines of P and T operations, using an evolutionary algorithm. It also makes use of gating and skip-connection mechanisms in the P and T operations, respectively. Section title: 5.3 Graph neural architecture search Educational score: 1.6633687019348145 Domain: other Document type: Other Language: en The search space includes P-T combinations and the number of P-T operations. The output of node v in the l-th layer is represented by o v ( l ) in a single P or T operation within a single GNN layer of the model. The layer indices of all P and T operations are included in two sets, L P and L T . The connections of P and T are depicted in Figure 1B and also described below. Section title: 5.3.1 Propagation connections Educational score: 1.712887167930603 Domain: other Document type: Other Language: en An imminent problem in GNNs is over-smoothing or under-smoothing, a problem that arises with too many or too few propagation operations. To achieve suitable smoothness for different nodes, the P operations are amplified with a gating mechanism. If the next operation is also P, the result of the l-th P operation is the propagated node embedding of o ( l −1) . On the other hand, if T is the next operation, a node-adaptive combination weight is allocated for the node embeddings propagated by all of the previous P operations. Formulatively: Section title: 5.3.1 Propagation connections Educational score: 2.460505485534668 Domain: other Document type: Other Language: en where a i = σ ( s · o v i ) represents the weight for the i-th layer output of node v . Here, s is the learnable vector shared among the entirety of nodes, and σ denotes the Sigmoid function. To ensure proper scaling, the Softmax function is employed to normalize the sum of gating scores, making it equal to 1. Section title: 5.3.2 Transformation connections Educational score: 1.5157456398010254 Domain: other Document type: Other Language: en An imminent issue with GNNs is the model degradation issue, caused by a hyperbolic amount of transformation operations and may result in a reduction of the model's accuracy. To mitigate this issue, skip-connection mechanisms are used in T operations. Each T operation's input is the total of all the T operations' outputs up to the last layer and the output from the layer before it. The input and output of the l-th T operation can be formulated as: Section title: 5.3.2 Transformation connections Educational score: 1.5875729322433472 Domain: other Document type: Other Language: en where m ( l ) represents the index of the last T operation before the l-th layer, and W ( l ) denotes the trainable parameter in the l-th T operation. Section title: 5.3.2 Transformation connections Educational score: 2.6904642581939697 Domain: biomedical Document type: Other Language: en Evolutionary algorithms are a class of optimization algorithms inspired by biological evolution that aim to achieve the best accuracy in offspring through mutations. In our case, each GNN architecture is represented as a sequence of P and T operations. Each pipeline can be considered a chromosome and the mutations that occur simulate nature's mutations. These mutations can happen at any random position in the sequence. In our instance, four different cases of mutation can be enforced: Section title: 5.3.2 Transformation connections Educational score: 2.974902629852295 Domain: biomedical Document type: Study Language: en Initially, k distinct GNN designs are generated at random and evaluated on the validation set. These architectures represent the initial population set Q. Subsequently, m (m < k) members of the population are randomly sampled, and parent A is determined by selecting the member with the highest validation accuracy. By enforcing a random mutation of the four presented on A, a child architecture B is produced. B is then evaluated and added to the population, and the oldest person is eliminated. After T generations of this procedure, the architecture with the best performance is eventually returned. Section title: 5.3.2 Transformation connections Educational score: 1.2120451927185059 Domain: other Document type: Other Language: en DFG-NAS returns a sequence of P and T steps. As illustrated in Figure 1A , each step consists of an RGCN that conducts one of the two main functions as we described incorporating both the user metadata and the user relations. After the RGCNs layers an MLP is employed to finally distinguish bots from genuine users. Section title: 6.1 Baselines Educational score: 1.1018271446228027 Domain: other Document type: Study Language: en We compare our proposed apporach to the state-of-the-art models that are referenced in the paper of BotRGCN . These experiments are all ran on the same dataset as the one we used for a fair comparison. We are using the published results for the comparison. More specifically, we compare our model to these state-of-the-art models: Section title: 6.2 Experiment settings Educational score: 1.673258662223816 Domain: other Document type: Other Language: en The experiment was run on Google Colab using Nvidia's T4 GPUs. The population set k for the architectural search is 15, and the maximum generation time T is 80. The training budget of each GNN architecture is 70 epochs. These numbers although limited due to our resources, provide a great example of the efficiency of our model. More complex architectures that we tested do not necessarily provide better results. Also, the number of epochs is sufficient to get a good idea of each architecture's accuracy. Adam optimizer is used for training, and its learning rate is set to 0.04. The criterion is Cross Entropy Loss and the regularization factor is 2e-4. Dropout is applied to all feature vectors at a rate of 0.5, and dropout among GNN layers is set to 0.8. Section title: 6.2 Experiment settings Educational score: 1.84938645362854 Domain: other Document type: Study Language: en After running the NAS method we process the results and examine the five architectures with the best accuracy in the validation set. Each architecture is now trained with 100 epochs on the TwiBot-20 dataset . The train set is 70% of the dataset, the validation set is 20% and the test set is 10%. Adam optimizer with a learning rate of 1e-3 is also used for training. Then each architecture is tested on the test set. We will present the findings of these experiments below. Section title: 6.3 Evaluation metrics Educational score: 1.5659897327423096 Domain: other Document type: Study Language: en We assess our model's performance using its Accuracy, F1-score, Precision, Recall, Specificity, and MCC. These metrics are computed by labeling the bots as the positive class and the genuine users as the negative class. To compare the performance of our model to the other baseline models we will only use the metrics Accuracy, F1-score, and MCC. Section title: 7 Results Educational score: 1.5367690324783325 Domain: other Document type: Other Language: en Each architecture during the search is saved with its P-T configuration, accuracy in the validation set, and accuracy in the test set. In Figure 2 , the five architectures with the highest validation accuracy that are chosen from the NAS method are depicted. Section title: 7 Results Educational score: 1.272925615310669 Domain: other Document type: Other Language: en These architectures are trained and tested from scratch in TwiBot-20 dataset. We present all the metrics attained by all the architectures in Table 4 . Section title: 7 Results Educational score: 1.2417021989822388 Domain: other Document type: Other Language: en All selections achieve good metrics and present advantages in bot detection over state-of-the-art methods. These results underscore the significant advantages that emerge from employing architecture search techniques regarding the field of bot recognition. Moreover, they establish the efficiency of utilizing user features and relationships between users in bot detection. Section title: 7 Results Educational score: 1.492326021194458 Domain: other Document type: Other Language: en Upon closer examination of the results, the third architecture achieves the best evaluation metrics. The fifth architecture has the highest precision. However, all the architectures present high metrics of accuracy, F1-score, and MCC and whichever architecture we choose could compete with state-of-the-art models. From now on we will refer to the third architecture as our model, since it provides the highest accuracy. Section title: 7 Results Educational score: 1.6423512697219849 Domain: other Document type: Study Language: en In Table 5 we present the performance of the baseline methods on the TwiBot-20 dataset compared to ours. We see that our model benefits from the search for the fittest architecture that we performed beforehand, as it achieves a higher accuracy, F1-score, and MCC than other state-of-the-art methods. Section title: 8 Ablation study Educational score: 1.3832539319992065 Domain: other Document type: Other Language: en To demonstrate our model's effectiveness and integrity we will perform an ablation study on the basic ideas: the user's features used for the training, the Gate operation, and the skip-connection operation. Section title: 8 Ablation study Educational score: 1.5368646383285522 Domain: other Document type: Study Language: en To prove that using multi-modal information is vital to our model performance we will train the architecture that produces the best results with reduced features. We will reduce one feature at a time and use combinations of the features for the training. We present the results in Table 6 . Section title: 8 Ablation study Educational score: 2.1202728748321533 Domain: other Document type: Study Language: en We see that training with reduced features may achieve higher metrics in some cases. Notably, training without descriptions has a higher F1-score than the original model but has a lower precision. Also, training without tweets has a higher recall value. Training without numerical properties has a higher precision and specificity but a lower MCC than training without description. Training with only the categorical and numerical properties has the highest recall. Therefore, training with combinations of features does not achieve as high metrics as training with all the features in each case, meaning that all features contribute to the model's performance. These remarks are important to consider for future research in ensuring the dataset's quality, but training the model with all the features provided makes it more adaptable to other datasets. For further understanding we will train the model using only one feature at a time, to investigate their importance separately. We present the results in Table 7 . Section title: 8 Ablation study Educational score: 2.4711992740631104 Domain: biomedical Document type: Study Language: en Obviously, the model trained with all the features has the best performance. From the results, we deduce that the categorical property is the feature that contributes the most to the model's sufficient accuracy. This ablation study proves that all features are advantageous for training our model to perform well in the task of bot detection. However, they do not contribute equally, and more studies to enhance the quality of the datasets could benefit future studies of bot detection. Section title: 8 Ablation study Educational score: 3.370131731033325 Domain: biomedical Document type: Study Language: en Next, we compare the architecture that results from the architecture search with a Gate operation and without a Gate operation. The findings of this ablation study are depicted in Table 8 . We see that the architecture without the gate has a reduced accuracy by 0.5% compared to the model's and a reduced F1-score by 0.46%. The gating mechanism dynamically consolidates information from all propagation steps, effectively regulating the smoothness of various nodes. Without it, the T operations take as input only the last output of the P steps. This is the reason the model underperforms without the Gate operation in the P functions, as it may suffer from over-smoothing. The architectures that are examined during this search have more T steps and shallower propagation processes, failing to obtain information from nodes during message passing as successfully as the original model. This ablation study proves the importance of the Gate operation in the P functions during our architecture search. Section title: 8 Ablation study Educational score: 1.9176274538040161 Domain: other Document type: Study Language: en Finally, we compare the architecture that results from the architecture search with a skip-connection operation and without a skip-connection operation. The findings of this ablation study are depicted in Table 9 . We see that the architecture without the gate has a reduced accuracy by 0.93% compared to the model's and a reduced F1-score by 1.2%. Without the skip-connection operation, the input of the T steps is only the output of the last step. This may lead to the degradation of the model as the transformation functions can increase. The processing of the messages from nodes is not as effective and the accuracy declines. This ablation study proves the importance of the skip-connection operation in the T functions during our architecture search. Section title: 9.1 Implications Educational score: 1.5042214393615723 Domain: other Document type: Study Language: en The proliferation of social media bots has prompted concerns regarding user safety and their broader societal impact. Bot detection, a focal point of contemporary studies, is not only explored through the lens of machine learning but also delves into the realms of social science. Various methodologies have been employed, encompassing supervised or unsupervised learning or a hybrid of both. A relatively recent and innovative approach involves Graph Neural Network (GNN)-based architectures, integrating diverse user features and interactions to construct a comprehensive graph representation. In our work, we formulate a heterogeneous graph that captures the following relationships between users, incorporating nodes with information on user profiles, tweets, and interests. This novel contribution enhances existing bot detection research by demonstrating the efficacy of integrating and analyzing user relationships. Section title: 9.1 Implications Educational score: 3.8259971141815186 Domain: biomedical Document type: Study Language: en As technology advances, the adaptive nature of bots poses an ongoing challenge for detection models, rendering many state-of-the-art architectures ineffective against newer datasets. The pressing need for adaptable models underscores the importance of overcoming the limitations associated with fixed architectures. Neural Architecture Search (NAS) models prove to be a promising solution, demonstrating their potential to enhance model efficiency in real-world tasks by automatically searching through various architectures. Historically, the adoption of NAS techniques for bot detection is limited, so we propose the implementation of an adapted DFG-NAS. By integrating DFG-NAS and tailoring it to Relational Graph Convolutional Networks, we explore optimal permutations of Propagation and Transformation steps in the message-passing protocol of the RGCN layers. Our investigation showcases superior performances of the top architectures compared to state-of-the-art models. Our work is one of the starting points in implementing architecture search models on bot detection. Our research findings encourage further exploration into how NAS models can automatically construct more effective architectures, resulting in a future restraint of the existence of bots. Section title: 9.2 Applicability of our approach to different types of social interaction Educational score: 0.7995413541793823 Domain: other Document type: Other Language: en In this section, we examine the applicability of our introduced approach to other types of social interaction besides social media. Section title: 9.3 Limitations Educational score: 2.119757652282715 Domain: other Document type: Study Language: en Our study comes with some limitations. Firstly, we conducted our experiments only on one dataset, which does not ensure generalizabilty of our proposed approach. Therefore, in the future, we aim to test our method on TwiBot-22 dataset . Secondly, our method is based on the collection of labeled data. Obtaining labeled data is a difficult task. For this reason, unsupervised and self-supervised learning algorithms have been developed for addressing the issue of labels' scarcity. Applying unsupervised and self-supervised learning in conjunction with our approach is one of our future plans. Thirdly, we did not tune the hyperparameters due to limited access to GPU resources. Hyperparameter tuning ensures that optimal performance is obtained. Finally, we represented each user as a concatenation of features. Concatenation does not capture the inherent correlation of the different modalities. In the future, we aim to use multimodal fusion methods for constructing each user's representation . Section title: 10 Conclusions and future work Educational score: 3.3522088527679443 Domain: other Document type: Study Language: en As social media continues to play a pivotal role in shaping public opinion and discourse, the development of effective and adaptive bot detection methods becomes increasingly crucial for maintaining the integrity and trustworthiness of online information. In this study, we introduced a novel model for identifying bots, integrating GNNs and NAS algorithms, demonstrating significant performance gains. The integration of Graph Neural Architecture Search empowered us to dynamically determine optimal combinations of propagation and transformation operations in the graph neural network architecture. This adaptive architecture effectively addresses the constraints imposed by fixed structures, introducing a level of flexibility essential for improving the performance on the bot detection task. From the experiment results we conclude that the five architectures with the highest validation accuracy, during the architecture search, are quite efficient in our task and compete with other models. Meanwhile, the one with the highest accuracy achieves a test accuracy of 85.68%, surpassing other state-of-the-art models for bot detection. The outcomes of the experiment present promising prospects for integrating more Neural Architecture Search (NAS) methods into the domain of bot detection in various social media platforms. Section title: 10 Conclusions and future work Educational score: 1.5228883028030396 Domain: other Document type: Other Language: en The exploration of dynamic graph adaptations stands as a crucial avenue for future research in the task of bot identification in social media platform X. The dynamic nature of social networks, characterized by the continuous incorporation of new users, necessitates the development of mechanisms to seamlessly integrate these additions into the evolving graph structure. Investigating methods for real-time graph updates and exploring how the model adapts to the inclusion of new users will enhance the system's agility in capturing emerging bot behaviors within the dynamic social landscape. Furthermore, the prospect of transferring our model to other social media platforms emerges as a key future avenue. Extending the applicability of our approach beyond X involves understanding the unique dynamics and characteristics of different platforms. Future work should focus on developing a transferable framework capable of recognizing bot-like behaviors across diverse social networks. By addressing the nuances and variations in user interactions and content features, we can contribute to the development of a versatile bot detection system with broader applications in the ever-expanding realm of social media platforms. | Other | other | en | 0.999998 |
PMC11695288 | Section title: Introduction Educational score: 4.058194160461426 Domain: biomedical Document type: Study Language: en Aflatoxin B1 (AFB1) are toxic secondary metabolites produced during the growth of molds and commonly found in grains and animal feed ( 1 – 3 ). AFB1 exposure may reduce feed intake and weight gain, disrupt rumen microbiota balance, impair liver and kidney function, and negatively affect overall health ( 4 , 5 ). Additionally, AFB1 can be transferred into animal products (meat and milk), thereby posing a risk to human health ( 6 , 7 ). A recent review by Eskola et al. ( 8 ) suggested that about 60 to 80% of the global food crops are contaminated with mycotoxins. In the subtropical region, the highest concentration was 3.76 mg/kg ( 9 ). Ma et al. ( 10 ) collected 742 feed ingredients samples from various regions of China. Among them, more than 83.3% of the samples was contaminated AFB1 at different concentrations. The highest concentration in China exceeded the standard of 1.6 mg/kg ( 9 ). Previous studies had shown that rumen microbiota have the ability to remove AFB1 ( 7 , 11 , 12 ), but the ability is limited. Therefore effective methods of enhancing the removal capacity of the original rumen microbiota for AFB1 are necessary to be developed. Section title: Introduction Educational score: 4.139301300048828 Domain: biomedical Document type: Study Language: en Regarding the food safety, dietary interventions with plant-derived additives are a promising approach to promoting the removal of AFB1 by rumen microbiota. In mammals, Indoleacetic-3-acid (IAA) is an important indole-derivative catabolized from dietary tryptophan by the intestinal microbiota, but intestinal dysbiosis can influence IAA production ( 13 ). IAA has various special functions in microbiota metabolism ( 14 , 15 ), and has a certain regulatory effect on the synthesis of tryptophan and cytochromes ( 16 ). Cytochrome CYP can oxidize AFB1, forming AFB1-8, 9-epoxide ( 17 , 18 ). Moreover, IAA was able to restore the intestinal microbiota balance and maintain its stability ( 19 ). But there are few reports on the removal of rumen microbiota and AFB1 by IAA. The objective of the present study was therefore to evaluate different dose of IAA in the rumen effect of the removal rate, ruminal fermentation profile, enzyme activity, and microbiota in vitro . Section title: Experimental design, procedure, and sampling Educational score: 4.11518669128418 Domain: biomedical Document type: Study Language: en Experiment 1: They were divided into 80 bottles and allocated to two groups: (1) the CK group, blank control group without AFB1; (2) the AFB1 group, with 1 mg/kg AFB1. The in vitro fermentation was independently conducted seven times for 0, 1, 2, 3, 4, 5, 24, and 48 h. Experiment 2: The removal rate of AFB1 in Experiment 1 was used to select 24 h as the fermentation time for Experiment 2. They were divided into 20 bottles and allocated to five groups: (1) the AFB1 group, with 1 mg/kg AFB1; (2) 1 mg/kg AFB1 + 15 mg/kg IAA group; (3) 1 mg/kg AFB1 + 150 mg/kg IAA group; (4) 1 mg/kg AFB1 + 1,500 mg/kg IAA group; (5) 1 mg/kg AFB1 + 7,500 mg/kg IAA group. Each treatment was performed inquintuplicate. The fermentation liquid was collected and stored in liquid nitrogen, pre-column derivatization ( 20 ) was carried out to detect AFB1 content and rumen fermentation indicators. Section title: Experimental design, procedure, and sampling Educational score: 4.129190444946289 Domain: biomedical Document type: Study Language: en In vitro ruminal fermentation followed the method described by Longland et al. ( 21 ). The rumen fluid was collected from three Holstein dry cows (650 kg ± 20 kg) before the morning feeding. The ingredients and chemical composition of total mixed ration are presented in Table 1 . The mixture of the rumen fluid was filtered by four layers of gauze, mixed with buffer solution (v/v = 1:1), and kept at 39°C in a water bath while continually flushed with CO 2 . A total of 0.2 g alfalfa silage (DM, 364.20 g·kg −1 FM; CP, 173.2 g·kg −1 DM; NDF, 389.5 g·kg −1 DM; ADF, 285.5 g·kg −1 DM) and 0.2 g starch (DM, 85.24 g·kg −1 FM; Starch, 612.1 g·kg −1 DM; CP, 55.8 g·kg −1 DM; NDF, 87 g·kg −1 DM; ADF, 21.9 g·kg −1 DM) as the fermentation substrate and placed in nylon bags. Afterwards, 60 mL of the diluted rumen fluid was divided into individual fermentation bottles, sealed, and placed in a shaker (120 rpm) at 39°C ( 22 ). Upon at 24 h, it was immediately removed, stopped in an ice bath, and samples were taken for detection of various indicators. Section title: Fermentation indicators, AFB1, fiber-degrading enzyme activity, and qPCR Educational score: 4.091518878936768 Domain: biomedical Document type: Study Language: en A total 1 mL of the collected rumen fluid was subjected to pre-column derivatization ( 20 ). The content of AFB1 was determined using an Agilent 1,260 Infinity II high-performance liquid chromatograph (HPLC) with a C18 column, 4.6 × 250 nm, 5 μm. The mobile phase was acetonitrile-water (20:80); column temperature: 40°C; mobile phase flow rate: 1.0 mL/min; injection volume: 20 μL; excitation wavelength 360 nm, emission wavelength 440 nm; detection time 20 min. The degradation rate of AFB1 = (AFB1 content in the blank control - AFB1 content in the experimental group)/AFB1 content in the blank control × 100%. Section title: Fermentation indicators, AFB1, fiber-degrading enzyme activity, and qPCR Educational score: 4.1066436767578125 Domain: biomedical Document type: Study Language: en pH was measured using a pH meter (LE438, Mettler Toledo Instruments Co., Ltd. China). For the other analyses, 25% meta-phosphoric acid was added to the fermentation fluid (1/5, v / v ), and then samples were centrifuged for 10 min at 10,000× g at 4°C using a high-speed freezing centrifuge . The supernatant was collected and stored at −80°C for NH 3 -N and VFA determinations. The content of ammonia nitrogen (NH 3 -N) was determined using the phenol-hypochlorous acid colorimetric method ( 23 ). The content of volatile fatty acids (VFA) (Acetic acid, Propionic acid, Butyric acid, Isobutyric acid, Valeric acid and Isovaleric acid) was determined using a high-performance gas chromatograph ( 24 ). The determination of the four types of cellulase (Carboxymethyl cellulase, Pectinase, Xylanase, α -glicosidase) was carried out according to the method described by Agarwal et al. ( 25 ). Section title: Fermentation indicators, AFB1, fiber-degrading enzyme activity, and qPCR Educational score: 4.10944128036499 Domain: biomedical Document type: Study Language: en Ruminal microbial DNA was extracted according to the method of Edrington TS ( 26 ). qPCR (primers were listed in Table 2 ) was used to determine the relative abundance of 10 bacteriain the incubation fluid. Real-time PCR was carried out on an Applied Biosystems Step One Plus Fast Real-Time PCR System (Applied Biosystems Co., USA). The reaction mixture (20 μL) were mixed with SYBR Premix Taq TM (10 μL, TaKaRa Biotechnology Co., Ltd., Dalian, China), ddH 2 O (7.0 μL), PCR forward or reverse primer (0.2 μmol L −1 , 0.8 μL), ROX Reference Dye (0.4 μL, 50×) and the template DNA (1 μL). The number of cycles required to reach a threshold adjusted for each taxon (Ct) was recorded for eachsample. The PCR programs included initial denaturation (1 cycle of 50°C for 2 min and 95°C for 2 min), primer annealing and product elongation [40 cycles of 95°C for 15 s and 60°C for 1 min ( 27 , 28 )]. Section title: Statistical analyses Educational score: 3.6771719455718994 Domain: biomedical Document type: Study Language: en Data of experimental 1 using Statistical Analysis System (paired sample T -test). Data of experimental 2 using the general linear model (GLM) procedure of Statistical Analysis System . The repeated measures model accounted for the fixed effects at different level of treatment. Tukey’s test was used for the multiple comparisons among mean values and linear and quadratic effects were calculated at p < 0.05, (* p < 0.05; ** p < 0.001). p < 0.05 was accepted as statistically significant, and p -values between 0.05 and 0.10 were considered to represent a statistical trend. Figures were drawn using KingDrawPc V3.0.2.20 (Qingdao Qingyuan Precision Agriculture Technology Co., Ltd., Qingdao, China), GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA), and Adobe Illustrator 2022–26.0 (Adobe Systems, San Jose, CA, USA) for graphic illustration. Section title: Interaction between AFB1 and rumen fermentation Educational score: 4.091917037963867 Domain: biomedical Document type: Study Language: en The removal efficiency of AFB1 exhibited a stable and significant increasing trend with the extension of incubation time . The removal rate of AFB1 increased rapidly in the first 24 h, after which the rate of removal slowed down between until 48 h. At 48 h, the removal rate of AFB1 can reach 80.09%. Considering the factors of removal efficiency, this experiment selects 24 h as the final fermentation time. Compared to the control group, AFB1 exposure significantly reduced the content of acetic acid ( p < 0.05); meanwhile, the content of propionic acid, isobutyric acid valeric acid, and isovaleric acid significantly increased ( p < 0.05). Further analysis shows that AFB1 exposure led to a significant decrease in the acetic acid/propionic acid ratio ( p < 0.05). In addition, AFB1 exposure did not significantly affect the pH value and NH 3 -N content of the in vitro fermentation fluid. Section title: Interaction between AFB1 and rumen fermentation Educational score: 3.8382604122161865 Domain: biomedical Document type: Study Language: en Compared to the control group, AFB1 exposure has a significant ( p < 0.05) effect on reducing the activity of xylanase, while it does not significantly affect the activity of pectinase, carboxymethyl cellulase and α -glucosidase . Section title: Interaction between AFB1 and rumen fermentation Educational score: 4.015929222106934 Domain: biomedical Document type: Study Language: en As shown in Figure 3 , the relative proportions of Prevotella ruminicola and Fusobacterium succinogenes significantly ( p < 0.05) decreased after AFB1 exposure, indicating that the presence of AFB1 inhibits the growth of these two types of bacteria. Section title: Effect of IAA supplementation on rumen fermentation and AFB1 removal Educational score: 3.3396666049957275 Domain: biomedical Document type: Study Language: en It can be observed that the content of IAA added showed a positive correlation with the removal rate of AFB1 . At concentration of 7,500 mg/kg IAA, AFB1 removal rate reached a maximum of 75.1%. Section title: Effect of IAA supplementation on rumen fermentation and AFB1 removal Educational score: 4.124300956726074 Domain: biomedical Document type: Study Language: en IAA at concentrations of 15 mg/kg and 150 mg/kg showed a positive correlation with the content of volatile fatty acids. Specifically, IAA at concentrations of 15 mg/kg and 150 mg/kg had a significant increasing trend in the content of acetic acid ( P L = 0.001), propionic acid ( P L = 0.012), butyric acid ( P L = 0.005), isobutyric acid ( P Q < 0.001), and valeric adid ( P L = 0.002), isovaleric acid ( P Q = 0.001) and total volatile fatty acids (TVFA) ( P L = 0.031). Furthermore, at 1500 mg/kg IAA, the content of acetic acid ( P L = 0.001), valeric acid ( P L = 0.002), the ratio of acetic acid/propionic acid ( P L = 0.001), and TVFA ( P L = 0.031) in the fermentation broth significantly increased, but the content of propionic acid ( P L = 0.012), butyric acid ( P L = 0.005), isobutyric acid ( P Q < 0.001), and isovaleric acid ( P Q = 0.001) significantly decreased trend. The addition of IAA did not significantly affect the pH and NH 3 -N content in the fermentation fluid . Section title: Effect of IAA supplementation on rumen fermentation and AFB1 removal Educational score: 4.112891674041748 Domain: biomedical Document type: Study Language: en The impact of adding different concentrations of IAA on the activity of major fiber-degrading enzymes in vitro fermentation fluid is shown in Figure 6 . A total of 15 and 150 mg/kg IAA significantly increased the activity of xylanase ( p < 0.05) and showed an enhancing trend for pectinase ( P L = 0.002) and carboxymethyl cellulase ( P Q = 0.005). In contrast, 1,500 mg/kg concentrations of IAA significantly decreased the activity of pectinase ( p < 0.05), exhibited a certain inhibitory effect on the activity of xylanase and carboxymethyl cellulase. Section title: Effect of IAA supplementation on rumen fermentation and AFB1 removal Educational score: 4.108919143676758 Domain: biomedical Document type: Study Language: en That 15, 150 and 1,500 mg/kg of IAA were added to the fermentation fluid, there was an increasing trend in the relative proportions of total bacteria ( P L = 0.026), B. fibrisolvens ( P L = 0.019), R. albus ( P L = 0.014), R. amylophilsus ( P L = 0.012), total methanogenic archaea ( P L = 0.003), P. ruminicola ( P L = 0.046) and F. succinogenes ( P L = 0.003) . However, the relative proportions of R. flavefaciens had a downward trend ( P L = 0.059). Section title: Discussion Educational score: 4.426083087921143 Domain: biomedical Document type: Study Language: en Previous studies had found that some ruminal microbiota have the ability to removal AFB1 ( 26 , 29 ). A total 1 mg/kg of AFB1 can cause a disturbance in the rumen microbiota and reduce the abundances of Prevotella and P. butyrivibrio ( 30 ). But higher concentrations (2.5 mg/kg) of AFB1 not only alter rumen fermentation pattern but also compromise the safety of liver function ( 26 ). In our study, as the fermentation time increased, AFB1 exposure gradually decreased, reaching a stable state after a period of time. Especially when the fermentation time reached 48 h, the removal efficiency of AFB1 can reach about 80%. This phenomenon can be attributed to two key factors: (1) ruminal microbiota reduce the toxicity of AFB1 through adsorption; (2) enzymes secreted by ruminal microbiota can break down the structure of AFB1, converting it into weakly toxic substances. In addition, research has shown that AFB1, as a harmful secondary metabolite widely present in feed and its raw materials. AFB1 can disrupt the balance of microbiota in the rumen when ingested by ruminants and enter the rumen, leading to change the rumen microbiota and affecting the normal fermentation process of the rumen ( 31 , 32 ). In our study, the pH showed no significant changes, indicating that the in vitro fermentation test was normal. VFAs are the main source of energy for ruminants to obtain from feed ( 33 ), but the exposure of AFB1 has a significant inhibitory effect on the rumen microbiota, leading to decrease in the total volatile fatty acids (TVFA) and change in the fermentation type. This corresponds to previous studies by Cao et al. ( 30 ). In vitro fermentation experiments showed that after AFB1 exposure, the relative proportions of core microbiota in the rumen such as Prevotella and Fusobacterium decreased, these presented the similar results as acetic acid changes. In addition, AFB1 also reduced the activity of xylanase, consistent with the changes of B. fibrisolvens , while the activity of other enzymes such as pectinase did not change significantly, which may be related to the amount of AFB1 exposure. AFB1 exposure can lead to produce changes in pectase activity ( 34 ). Although the removal mechanism of AFB1 unclear, growing evidence has revealed that rumen microbiota dysbiosis is involved in the exposure of AFB1. Therefore, the restoration of rumen microbiota balance is likely to contribute to AFB1 removal. Section title: Discussion Educational score: 4.21561336517334 Domain: biomedical Document type: Study Language: en IAA is a crucial biological regulatory substance for the growth of plants, which is considered to be an effective and environmentally friendly additive with the potential to modulate the balance of animal microbiota. Although research on IAA in rumen microbiota is still in its infancy, this experiment has confirmed that adding different concentrations of IAA to rumen fluid containing AFB1 can effectively reduce the content of AFB1. IAA can activates the expression of cytochrome genes, such as cytochrome P450 enzymes have been found in different organisms, that is a key enzyme that involve in the biotransformation of AFB1 and affects its toxicity ( 17 ). Moreover, IAA can maintain microbiota homeostasis and improve microbiota disorders by activating signal transduction pathways through its specific ligands ( 35 ). Although the mechanism by which IAA removes AFB1 is not yet clear, a growing body of evidence suggests that the rumen microbiota is involved in the removal of AFB1. Therefore, we have focused on the metabolic mechanisms of rumen microbiota with the addition of IAA. Considering that high concentrations of IAA may affect the balance of the rumen, according to the “Safety Use Specifications for Feed Additives” the recommended amount of tryptophan (the precursor of IAA) in the diet of ruminants is 0.1% (1 g/kg). The dosage of IAA is based on previous reports (50 mg/kg body weight) ( 36 ) and our preliminary studies, and with a rumen volume of 50 L, it is considered safe for cattle. In addition, some studies have shown that excessively high concentrations (50 mg/kg body weight) of IAA may change the animal microbiota ( 19 ). Therefore, this experiment further explored the specific impact of IAA additions of 15, 150, and 1,500 mg/kg on rumen fermentation parameters, aimed to verify the addition of a reasonable range of IAA concentrations to mitigate the impact of AFB1 on the rumen fermentation process, thereby enhancing animal health. Section title: Discussion Educational score: 4.317453861236572 Domain: biomedical Document type: Study Language: en In our study, found that 150 and 15 mg/kg concentrations of IAA can significantly increase the content of TVFA, while 1,500 mg/kg concentrations of IAA tend to decrease. Indicate that the addition of IAA, enhances the rumen microbiota ability to utilize carbon sources and stimulates the degradation of nutrients within the rumen ( 37 ). In the experiment, after the addition of IAA, the content of acetic acid showed a linear upward trend, this is attributed to the metabolic fermentation products of R. amylophilus and P. ruminicola being primarily acetic acid ( 38 , 39 ). With the addition of IAA, the observed changes in the microbiota, such as Prevotella and Fusobacterium , are closely related to the adjustment of VFAs composition ( 40 , 41 ). These microbiota decompose the fiber in the feed, promoting the production of acetic acid and propionic acid. Propionic acid is key in the gluconeogenesis process, affecting body fat and lactose synthesis, this is primarily due to F. succinogene impact on the production of propionic acid through the propionate metabolism pathway ( 42 – 46 ). The concentration of IAA has a significant regulatory effect on propionic acid production, with 150 and 15 mg/kg concentrations of IAA causing a linear increase in propionic acid content, while 1,500 mg/kg concentrations of IAA reduce the level of propionic acid. F. succinogene also exhibited a trend of increasing first and then decreasing in this study. The change in butyric acid content is also related to the amount of IAA added, with 150 mg/kg and 15 mg/kg concentrations of IAA increasing butyric acid content, while 1,500 mg/kg concentrations of IAA reduce butyric acid content. B. fibrisolvens metabolize the production of butyric acid in rumen fluid, and the addition of 1,500 mg/kg IAA may make the microbiota rapidly metabolize and compete for rumen nutrients, resulting in the poor competitiveness of B. fibrisolvens and reducing the production of butyric acid ( 47 ). In addition, the regulation of the acetic acid/propionic acid ratio affects microbiota protein synthesis and the structure of the rumen microbiota, which relates to the digestion and nutritional metabolism of the whole body ( 48 , 49 ). In the rumen ecosystem, bacteria can degrade and utilize starch and plant cell wall polysaccharides, such as xylan and pectin. These bacteria play an important role in the degradation of protein and the absorption and fermentation of peptides ( 50 – 53 ). The addition of IAA has a significant impact on the changes in the rumen microbiota and the activity of the main fiber-degrading enzymes. A total of 15 and 150 mg/kg concentrations of IAA had increased trend of the activity of pectinase, xylanase, and carboxymethyl cellulase, while 1,500 mg/kg concentrations of IAA reduced the activity of these enzymes. These results show that IAA alleviates the imbalance of the rumen fermentation caused by AFB1 by regulating the rumen microbiota and enzyme activity. R. albus , B. fibrisolvens and R. flavefaciens , as the main fiber-degrading bacteria, can produce xylanase. In this study, the significant increase in R. albus and B. fibrisolvens is consistent with the changes in xylanase. The decrease in xylanase with the addition of 1,500 mg/kg concentrations of IAA may be related to the downward trend of R. flavefaciens . The changes in carboxymethyl cellulose are mainly caused by P. ruminicola , and pectinase follows the relative changes of R. flavefaciens and B. fibrisolvens ( 36 ). In summary, the appropriate addition of IAA had a positive impact on the rumen microbiota and metabolic products, but 1,500 mg/kg concentrations of IAA may inhibit the rumen fermentation process. These findings provide important information for optimizing rumen fermentation and improving the nutritional absorption of ruminants. Section title: Conclusion Educational score: 4.046648025512695 Domain: biomedical Document type: Study Language: en This experiment preliminarily explored the impact of in vitro addition of IAA on the removal rate of AFB1 and rumen fermentation. It was found that IAA in the range of 150 mg/kg addition could improve the removal rate of AFB1 and reduce the negative effects of AFB1 on in vitro fermentation characteristics and fermentation end-products. This provides a new strategy to mitigate the potential threat of AFB1 to animal health. | Study | biomedical | en | 0.999997 |
PMC11695289 | Section title: Introduction Educational score: 4.208648681640625 Domain: biomedical Document type: Clinical case Language: en Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary artery pressure and vascular resistance, leading to right heart failure and increased systemic venous pressure ( 1 ). This elevated pressure can extend to the cavernous sinus and ophthalmic veins, resulting in ocular complications such as central retinal vein occlusion (CRVO). Cilioretinal artery occlusion (CilRAO) is particularly intriguing pathophysiologically because it receives blood supply from the choroidal circulation rather than the retinal circulation ( 2 ). The combination of CilRAO and CRVO presents a unique clinical scenario that warrants thorough examination and discussion. This case report presents a novel instance of PAH-related CRVO leading to CilRAO, underscoring the intricate relationship between systemic cardiovascular conditions and ocular health. Section title: Case report Educational score: 2.6959478855133057 Domain: clinical Document type: Clinical case Language: en A 13-year-old girl was referred to our hospital in April 2023 with sudden, painless vision loss and a central visual field defect in her left eye persisting for 3 weeks. Her medical history included a surgically repaired ventricular septal defect (VSD) at age eight. She reported no significant personal or family medical history, and no prior use of medication. Section title: Case report Educational score: 4.145735740661621 Domain: clinical Document type: Clinical case Language: en Two weeks prior, an evaluation at a local hospital recorded a best-corrected visual acuity (BCVA) of 20/20 in the right eye and 20/50 in the left eye, with normal intraocular pressures (IOP) bilaterally. Fundus photography revealed pale gray-white retinal swelling above the fovea along the superior papillomacular bundle, flame-shaped hemorrhages, and blurred optic disc margins in the left eye, accompanied by significant retinal vein dilation and tortuosity . The right eye showed mild disc edema but was otherwise normal. Fluorescein angiography (FFA) locally showed several notable findings: delayed background fluorescence, delayed perfusion of the cilioretinal artery , hypofluorescence extending from the fovea to the superior vascular arcade in the posterior pole, as well as dilation and tortuosity of the veins with delayed venous filling . The right eye showed no evidence of stasis or fluorescein leakage on the optic disc, consistent with pseudopapilledema . Section title: Case report Educational score: 4.078238010406494 Domain: clinical Document type: Clinical case Language: en Upon admission, her BCVA was 20/50 in the left eye and 20/20 in the right. The ocular adnexa, anterior segment, and IOP were normal, as were pupillary light reflexes. Compared to prior evaluations at the local hospital, fundoscopic examination showed a reduction in the gray-white retinal edema above the fovea in the left eye, with no significant changes otherwise. FFA showed prolonged choroidal perfusion, delayed cilioretinal artery filling, scattered fluorescence blockage, widespread capillary dilation, and optic disc stasis in the late angiographic phase . The static visual field test recorded a sectoral scotoma adjacent to the fovea in the left eye . Spectral-domain optical coherence tomography (SD-OCT) revealed disorganization of the inner macular layers, corresponding to hyporeflective areas on near-infrared (NIR) imaging, with interspersed hyperreflective foci . Optic disc edema was confirmed with increased disc thickness and elevation . Based on the patient’s symptoms, clinical signs, and comprehensive ophthalmological examination, she was diagnosed with CilRAO combined with CRVO in the left eye. Section title: Case report Educational score: 3.4033286571502686 Domain: biomedical Document type: Clinical case Language: en Laboratory tests revealed mild anemia (hemoglobin: 92 g/L) and a slight elevation in platelet count (385 × 10^9/L). Other tests, including random blood sugar, serum C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), renal function, coagulation profile, antiphospholipid antibodies, homocysteine levels, lipid profile, and D-dimer, were all within normal ranges. Section title: Case report Educational score: 4.054909706115723 Domain: clinical Document type: Clinical case Language: en The etiology of CilRAO and CRVO was unclear based on initial findings. Given her VSD history, further investigations were recommended. Despite initial hesitation, the patient, who denied any symptoms beyond ocular complaints and requested immediate ophthalmic treatment, eventually agreed after counseling. Cardiothoracic consultation revealed mild enlargement of the right ventricle and right atrium on echocardiography. The atrial septum appeared intact, with an echogenic patch on the ventricular septum, confirming no shunt signal. Mild dilation of the pulmonary artery and a regurgitant jet in the right ventricular outflow tract were also noted. Tricuspid regurgitation was widely distributed within the right atrium during systole, with a continuous wave (CW) Doppler measurement estimating the pulmonary artery systolic pressure (PASP) at 80 mmHg. Duplex ultrasound of the carotid arteries showed smooth intimal surfaces bilaterally. A Transcranial Doppler (TCD) foaming test detected three emboli in the middle cerebral artery during a Valsalva maneuver, suggesting a potential right-to-left shunt. Head computed tomography (CT) and computed tomography angiography (CTA) of the cerebral and aortic arteries revealed no significant abnormalities. Section title: Case report Educational score: 2.5988714694976807 Domain: clinical Document type: Clinical case Language: en The patient was diagnosed with severe pulmonary arterial hypertension (PAH) during a cardiothoracic consultation and was prescribed Bosentan at a dose of 31.25 mg twice daily. She also received traditional Chinese herbal treatment to enhance ocular circulation. The decoction included Prunus persica (peach kernel), Carthamus tinctorius (safflower), Rehmannia glutinosa (raw Rehmannia root), Angelica sinensis (Dong Quai), Paeonia lactiflora (red peony root), Ligusticum chuanqiong (Szechuan lovage), and Achyranthes bidentata (ox knee), which are traditionally used to promote blood circulation and alleviate stasis. After 1 week of treatment, she requested discharge. Section title: Case report Educational score: 3.7716519832611084 Domain: clinical Document type: Clinical case Language: en At the one-month follow-up, the patient’s BCVA in the left eye improved to 20/32. Fundus photography showed reduced optic disc edema, venous tortuosity, and intraretinal hemorrhages. OCT revealed asymmetric thinning of the inner retinal layers in the macular region affected by CilRAO, along with substantial improvement in optic disc edema. Echocardiography showed a decrease in PASP to 65 mmHg. The cardiothoracic surgeon advised continuing oral Bosentan therapy. Section title: Case report Educational score: 3.9047129154205322 Domain: clinical Document type: Clinical case Language: en At 9 months, the patient’s left eye BCVA improved to 20/20. Fundoscopic examination revealed near-normal conditions . However, visual field testing detected a persistent sectoral scotoma adjacent to the macular fovea . OCT showed significant thinning of the inner retinal layers extending temporally and superiorly to the macular fovea , along with complete resolution of optic disc edema. Echocardiography indicated a PASP of 52 mmHg. Liver and kidney function tests were normal, and the patient continued oral Bosentan therapy. At the most recent follow-up, the patient’s left eye BCVA remained at 20/20, with no symptoms other than a paracentral scotoma. Her PASP remained stable at 45 mmHg with ongoing Bosentan treatment. Section title: Discussion Educational score: 3.99188494682312 Domain: biomedical Document type: Study Language: en Cilioretinal arteries are congenital vascular variants found in approximately one-third of normal eyes, originating from the peripapillary choroid or short posterior ciliary arteries. Their presence can be identified through clinical examination and FFA in about 20–32% of individuals ( 3 ). Unlike the central retinal artery, cilioretinal arteries lack autoregulatory mechanisms, making them more susceptible to hemodynamic changes and ischemic events ( 4 ). Section title: Discussion Educational score: 4.077342510223389 Domain: biomedical Document type: Study Language: en CilRAO is uncommon, comprising approximately 5% of all retinal artery occlusions. It can be categorized into three primary etiological groups: isolated CilRAO, CilRAO associated with CRVO, and CilRAO attributed to systemic autoimmune conditions such as giant cell arteritis ( 5–7 ). In this case, FFA revealed CRVO with delayed and sluggish flow in the cilioretinal artery rather than complete non-perfusion. Additionally, delayed background fluorescence suggested that the CilRAO was caused by hemodynamic disturbances—a high-resistance type of CilRAO associated with CRVO. Section title: Discussion Educational score: 4.304065227508545 Domain: biomedical Document type: Study Language: en The increased intraluminal pressure from CRVO likely led to secondary functional blockage of the cilioretinal artery due to its inability to autoregulate under elevated pressure conditions. The resulting hypoperfusion causes ischemia, particularly in the retinal tissue supplied by the cilioretinal artery. This pathophysiological mechanism aligns with literature suggesting that CRVO-induced elevated capillary pressure may lead to secondary arterial occlusions such as CilRAO ( 2 , 4 , 5 , 7 , 8 ). Section title: Discussion Educational score: 4.459439277648926 Domain: biomedical Document type: Study Language: en PAH is defined as a persistent pulmonary artery pressure exceeding 25 mmHg at rest or 30 mmHg during exercise ( 1 ). The pulmonary arterial (PA) tree, with its fractal branching structure, is designed to evenly distribute blood flow to the alveoli ( 1 ). However, conditions such as ventricular septal defect (VSD) that increase pulmonary blood flow can trigger neointimal remodeling, involving intimal hyperplasia, elastic lamina degradation, pericyte infiltration into the intima, and encroachment of smooth muscle cells into the lumen. This remodeling is driven by an imbalance between cell proliferation and apoptosis, upregulation of anti-apoptotic signaling, and persistent inflammation, ultimately causing thickening and fibrosis of the pulmonary artery walls, reduced luminal diameter, and increased vascular resistance ( 9–11 ). Section title: Discussion Educational score: 4.2227325439453125 Domain: biomedical Document type: Study Language: en While surgical repair of VSD may immediately correct abnormal blood flow, the pre-existing vascular remodeling often persists due to prior exposure to prolonged shear stress and turbulent flow, maintaining elevated pulmonary artery pressures and contributing to ongoing PAH ( 10 , 11 ). The timing of VSD repair is crucial, as delays can result in irreversible vascular remodeling and permanent pulmonary hypertension ( 1 , 12 ). As to this case, late surgical correction likely contributed to significant vascular changes and sustained pulmonary hypertension, despite structural defect resolution ( 13 ). Section title: Discussion Educational score: 4.178437232971191 Domain: biomedical Document type: Study Language: en PAH can elevate pressure in the superior vena cava, internal jugular vein, cavernous sinus, and ultimately the ophthalmic veins. The elevated pulmonary artery pressure leads to retrograde pressure affecting the right ventricle, right atrium, superior vena cava, and cavernous sinus, disrupting venous outflow from the eye and resulting in CRVO ( 14 ). In this patient, echocardiography revealed signs of severe PAH, including right ventricular and atrial enlargement, tricuspid regurgitation, and elevated pulmonary artery pressures, confirming the systemic contribution to the observed CRVO. Section title: Discussion Educational score: 4.249431610107422 Domain: biomedical Document type: Clinical case Language: en The management of this case emphasizes the importance of treating the primary disease, PAH, to alleviate its secondary ocular complications. Bosentan, an endothelin receptor antagonist, plays a pivotal role in lowering pulmonary artery pressure by blocking endothelin-1 (ET-1) receptors on vascular smooth muscle cells, leading to vasodilation and decreased vascular resistance. This reduction in pulmonary artery pressure decreases the retrograde venous pressure transmitted to the ophthalmic veins, thereby alleviating CRVO. By improving pulmonary hemodynamics, Bosentan reduces the venous congestion in the retinal circulation, facilitating the restoration of normal venous outflow. Furthermore, the normalization of retinal venous pressure helps reverse the hemodynamic imbalance causing the CilRAO. Since the CilRAO in this case is hemodynamically induced rather than due to an embolic event, lowering the venous pressure allows for improved perfusion in the cilioretinal artery territory. Although the patient’s central vision may improve with the resolution of CRVO and the reversal of CilRAO, ischemic damage caused by the initial hypoperfusion may leave lasting effects, such as paracentral scotomas. Section title: Discussion Educational score: 3.880591869354248 Domain: biomedical Document type: Other Language: en For elderly patients with combined CilRAO and CRVO, atherosclerosis, thrombophilia, vasculitis, and autoimmune conditions should be assessed. In younger patients, rarer etiologies, including elevated homocysteine levels, syphilis, patent foramen ovale, and HELLP syndrome, must be considered ( 4 ). In this case, detailed patient history revealed a past VSD repair, underscoring the need to thoroughly investigate medical history in such instances. Section title: Discussion Educational score: 3.7259280681610107 Domain: biomedical Document type: Clinical case Language: en This case represents the first reported instance of CRVO combined with CilRAO and PAMM as initial manifestations of PAH. It underscores the interplay between systemic vascular diseases and ocular health and emphasizes the importance of addressing systemic causes to achieve favorable ophthalmic outcomes. Section title: Conclusion Educational score: 4.108158111572266 Domain: clinical Document type: Clinical case Language: en The case we presented here underscores the intricate relationship between systemic cardiovascular conditions and ocular health, highlighting the first reported instance of CRVO combined with CilRAO secondary to PAH. Managing such cases emphasizes the importance of addressing the primary disease, PAH, to alleviate secondary ocular complications and prevent severe systemic issues. Bosentan, an endothelin receptor antagonist, played a pivotal role in lowering pulmonary artery pressure, reducing venous pressure, and alleviating CRVO. This approach addresses the root cause of increased retinal venous pressure, providing a comprehensive treatment strategy. Early intervention and holistic treatment are crucial in preventing permanent vision damage and serious systemic complications. This case highlights the need for clinicians to remain vigilant about systemic conditions that can impact ocular health and to adopt a multidisciplinary approach in managing complex cases. | Clinical case | biomedical | en | 0.999997 |
PMC11695290 | Section title: Introduction Educational score: 4.027886390686035 Domain: biomedical Document type: Study Language: en Chinese mitten crabs ( Eriocheir sinensis ) are a valuable aquaculture species in East Asia . However, the expansion of intensive aquaculture models has created new opportunities for the crab farming industry but has also led to frequent occurrences of bacterial , viral , and parasitic diseases . These diseases have resulted in significant decreases in farm production and substantial economic losses Although bacterial diseases in crabs are less common than viral and protozoan diseases , research has primarily focused on marine crabs that develop bacteremia or bacterial diseases affecting the exoskeleton . Previous studies on Callinectes sapidus , Callinectes bocourti , and Cancer irroratus have observed significant reductions in blood cell counts and intravascular coagulation following Vibrio infection, with endotoxins in the bacterial cell wall penetrating tissues . Several studies have demonstrated the ability of Vibrio parahaemolyticus (V. parahaemolyticus) to infect Chinese mitten crabs and their offspring . Further research on the infection process and the harm caused by V. parahaemolyticus to Chinese mitten crabs is crucial for enhancing the overall health of crab breeding practices. Section title: Introduction Educational score: 4.342728614807129 Domain: biomedical Document type: Study Language: en The extensive range of genes and virulence factors of Vibrio splays a crucial role in its heightened pathogenicity, which allows it to successfully invade a variety of host species, causing severe damage while evading host defenses. One highly dangerous species is V. parahaemolyticus , which poses a threat to both humans and aquatic animals in freshwater, estuarine, and marine environments . The genomes of V. parahaemolyticus exhibit a high level of diversity, with two chromosomes, frequent horizontal gene transfer, and high recombination rates . Toxigenic strains of V. parahaemolyticus produce various virulence factors, such as the heat-stable direct haemolysin (TDH) and the TDH-associated haemolysin, which are responsible for Kanagawa hemolysis. The encoding genes for these toxin factors are tdh and trh , respectively . Nonetheless, strains lacking hemolysins can also be associated with human infections . In the aquaculture process, we concentrate on specific strains that have the potential to cause diseases in aquatic animals. Section title: Introduction Educational score: 4.1546244621276855 Domain: biomedical Document type: Study Language: en V. parahaemolyticus has been expanding globally, with epidemics often linked to coastal warming . In 2021, a specific strain of V. parahaemolyticus emerged in Asia, primarily impacting crustaceans, particularly shrimp . Infection with this bacterium, referred to as acute hepatopancreatic necrosis disease (AHPND) or early mortality syndrome (EMS), has been extensively researched and found to carry the binary toxins PirA Vp and PirB Vp in the extrachromosomal virulence plasmid pVA1 . Clinical indications of AHPND in shrimp include a damaged hepatopancreas, soft shells, discontinuous or atrophic gut, absence of contents, and pale discoloration, often resulting in mortality rates as high as 100% . Our previous research has shown that this strain has the potential to infect freshwater crustaceans . Given that the Chinese mitten crab is a migratory and reproducing crustacean, it is also vulnerable to infection. Hence, it is imperative to conduct additional research on the infection mechanism and impact of V. parahaemolyticus on Chinese mitten crabs. Section title: Introduction Educational score: 4.200849533081055 Domain: biomedical Document type: Study Language: en The protection of multicellular organisms from invasive species has greatly relied on the evolution of the immune system. To resist the invasion of pathogenic microorganisms, two effective immune systems have evolved: innate immunity and adaptive immunity . Invertebrates exhibit limited adaptive immunity and primarily depend on innate immunity to combat pathogen invasion . E. sinensis , being an invertebrate, predominantly utilizes innate immune mechanisms, such as agglutination, encapsulation, phagocytosis, coagulation proteins, and bactericidally active peptides . The humoral immune system activates the phenol oxidase (ProPO) system, which leads to the production of antimicrobial peptides (AMPs). Cellular immune responses primarily involve phagocytosis and encapsulation of pathogenic bacteria . The current understanding of the immune regulation of Chinese mitten crabs in response to V. parahaemolyticus infection is limited. Further investigation is needed to explore the immune response of Chinese mitten crabs at various stages of Vibrio infection and analyze the differential transcription of the transcriptome. Section title: Introduction Educational score: 4.148380279541016 Domain: biomedical Document type: Study Language: en E. sinensis , a migratory crustacean, is susceptible to infection by Vibrio , particularly V. parahaemolyticus , which carries the priA / priB toxin gene. This study aims to develop an animal infection model using molecular biology and RNA-seq technologies. The model will be used to comprehensively analyze the changes in body fluid enzyme activity factors, hepatopancreas damage, and hepatopancreas function of E. sinensis during the infection process with V. parahaemolyticus . Furthermore, the study will investigate the differential expression of transcriptome genes, immune response, and changes in body indicators of experimental animals during the infection process. The findings from this study will contribute to a better understanding of the infection process of V. parahaemolyticus and the host’s immune mechanism. Section title: Information about test animals and V. parahaemolyticus Educational score: 4.043632507324219 Domain: biomedical Document type: Study Language: en Specimens of E. sinensis were acquired from Baodao Agricultural Technology Co., Ltd., located in Dongping County, Shandong Province, which specializes in aquaculture. These crabs had a mean weight of 15 ± 0.5 grams. They were housed in a laboratory tank equipped with a circulating water system and aerated with tap water. The temperature of the water was maintained at 26 ± 1.5°C. To provide hiding spots, PE pipes and artificial water plants were placed within the tank. Prior to the experiment, the crabs were acclimated in the laboratory for a duration of 1 week. Pre-experiments and acute infection experiments were conducted once the crabs’ state was stable. Prior to the commencement of the experiment, we randomly selected 10 experimental animals for the assessment of V. parahaemolyticus . The results indicated that these experimental animals did not harbor V. parahaemolyticus . Throughout the acclimation period, the crabs were provided with commercial crab feed; however, feeding was ceased on the day preceding the experiment. Specifically, the strain of V. parahaemolyticus was isolated from red claw crayfish culture ponds. Section title: Information about test animals and V. parahaemolyticus Educational score: 4.111577987670898 Domain: biomedical Document type: Study Language: en The V. parahaemolyticus strain utilized in this experiment was obtained, purified, and identified by the Research Laboratory of Aquatic Animal Health Breeding within the College of Animal Science and Technology at Shandong Agricultural University. This strain is stored at the Phage Research Centre within the College of Agriculture at Liaocheng University. The whole genome analysis of the strains utilized has been completed, and the original sequencing data are stored in the National Center for Biotechnology Information (NCBI) under the storage number PRJNA902323. To culture the strains, overnight growth was achieved on citrate bile salt sucrose agar sulphate plates . Single colonies were then transferred to LB broth and incubated for 6 hours. Subsequently, 1 mL of broth was centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and 50 μL of RNase-free water was added. To detect the virulence genes carried by the experimental strains, PCR was used. After amplification and sequencing of the PCR products, we performed BLAST comparative analysis on all valid PCR results using the NCBI database. The sequences of all virulence genes can be found in Table 1 . The primer sequences pertinent to this article are also listed in Table 1 . Section title: Modeling of infection and sample collection Educational score: 4.094432830810547 Domain: biomedical Document type: Study Language: en A total of 100 specimens of Chinese mitten crabs were randomly distributed into 5 groups, each comprising 20 individuals. Cultivated strains (100 mL) were incubated overnight at a temperature of 37°C, after which they were centrifuged at a speed of 4000 rpm for 10 minutes using a high-speed centrifuge. The supernatant was discarded, and the strains were washed three times using a phosphate buffer solution. The concentration of the bacterial solution was measured using an ultra-microspectrophotometer and subsequently diluted with a buffered salt solution to obtain concentrations of 1×10 5 , 1×10 6 , 1×10 7 , and 1×10 8 CFU/mL. Each experimental subject was administered an injection of 50 μL of the bacterial solution, whereas the control group received an injection of the same volume of phosphate-buffered saline (PBS). Section title: Modeling of infection and sample collection Educational score: 4.096294403076172 Domain: biomedical Document type: Study Language: en The mortality rate of the experimental group was recorded at various intervals following the initiation of the disease ( Supplementary Table S1 ), and the LD50 concentration of the pathogenic strain was determined to be 1.78×10 6 CFU/mL after 24 hours using this methodology . In the subsequent stage, 200 experimental subjects were arbitrarily selected and divided equally into two groups: the experimental group and the control group, each containing 100 specimens. The experimental group was administered an injection of 50 μL of fresh bacterial solution at the LD50 concentration, whereas the control group was administered an injection of the same volume of phosphate-buffered saline (PBS). Section title: Modeling of infection and sample collection Educational score: 4.1283721923828125 Domain: biomedical Document type: Study Language: en Samples of tissue from the hepatopancreas were procured at intervals of 6, 12, 24, 48, and 72 hours after the infection. Based on clinical symptoms and behavioral observations of Chinese mitten crabs, individuals exhibiting significant pathological conditions within 6 hours after challenge were categorized as the susceptible group (Es_SC), whereas those in good health or displaying no apparent symptoms after 24 hours were classified as the disease-resistant group (Es_AI). The individuals injected with PBS were designated as the control group (Es_CG). When collecting samples, begin by using 75% alcohol-soaked cotton balls to thoroughly wipe the entire body of the experimental animals. Once all samples have been obtained, they should be rapidly frozen in liquid nitrogen to preserve their freshness, and stored in a refrigerator at -80°C for subsequent assays to determine immunoenzymatic activity and extract RNA, respectively. Additionally, hepatopancreatic tissues were collected at different intervals after rinsing with phosphate-buffered saline and fixing them with a solution of 4% paraformaldehyde. Pathological sections were prepared to observe any changes in pathology . Section title: Histopathological analysis Educational score: 4.097636699676514 Domain: biomedical Document type: Study Language: en Histopathological analysis was conducted on the hepatopancreas of E. sinensis at 6, 24, and 72 hours after infection, with the PBS injection group serving as the control. The hepatopancreas was fixed with paraformaldehyde for 24 hours and then embedded in paraffin blocks. Subsequently, the tissue was dehydrated using different concentrations of ethanol. Using a microtome, the blocks were sectioned into thicknesses of 5-6 μm. The resulting sections were stained with hematoxylin and eosin and subsequently examined under light microscopy following fixation with resin. Section title: Detection of humoral immunoenzymatic activity Educational score: 4.1073198318481445 Domain: biomedical Document type: Study Language: en To detect humoral immunoenzymatic activity, tissue samples were thawed from the -80°C refrigerator. Filter paper was de-watered, weighed, and cut appropriately with scissors. The cut tissue samples were placed in sterile 1.5 mL centrifuge tubes and mechanically ground on ice using a grinder. After spinning at 2500 revolutions per minute (rpm) for 10 minutes, the liquid above was discarded. Subsequently, the tissue was mixed with saline solution to produce a 1% solution of crushed tissue for examining the activities of immune-related enzymes. The primary immune-related enzymes evaluated included acid phosphatase (ACP: A060-2-2), peroxidase (PO: A084-1-1), total antioxidant capacity (T-AOC: A015-2-1) alkaline phosphatase (AKP/ALP: A059-3-1), alanine transaminase (GPT/ALT: C009-1-1), aspartate transaminase (AST/GOT: C010-2-1), Lysozyme (LZM: A050-1-1) and superoxide dismutase (SOD: A001-4-1). These kits were procured from Nanjing Jiancheng Bioengineering Institute in China ( http://www.njjcbio.com/ ) and were used according to the manufacturer’s instructions. Three biological replicates were conducted for each sample. Section title: RNA sequencing grouping Educational score: 4.109027862548828 Domain: biomedical Document type: Study Language: en RNA was extracted from three samples randomly selected from a total of nine collected samples, which were maintained at low temperatures throughout the process. Total RNA extraction from crabs was performed according to the manufacturer’s instructions using TRIzol (Invitrogen, USA). Each high-quality, non-degraded RNA sample underwent library construction using the NEBNext ® Ultra™ RNA Library Preparation Kit (NEB, USA) with 1 μg per sample. The HiSeq 4000 platform (Illumina, USA) was used to generate 150 bp paired-end (PE) reads. RNA-Seq analysis and clustering were outsourced to Novogene Biotechnology Co., Ltd. in Beijing, China, which employs Illumina sequencing technology. Initial processing of raw data (raw reads) in fastq format was performed using an in-house perl script. Section title: Data quality control and bioinformatics analysis Educational score: 4.05192756652832 Domain: biomedical Document type: Study Language: en For RNA-seq data without a reference genome, data quality control was performed with the Trimmomatic software to remove adapters and low-quality reads (N content >5%, base mass values <20). Clean reads were obtained and then assembled using Trinity to generate a reference sequence for subsequent analysis. Section title: Data quality control and bioinformatics analysis Educational score: 4.119417190551758 Domain: biomedical Document type: Study Language: en To identify genes that are under-expressed or only partially detected across samples, all samples from the same species were combined to achieve a comprehensive assembly result for subsequent analyses, such as differential expression analysis. However, samples from different species underwent separate assemblies due to significant genomic differences. In the absence of a reference genome, transcriptome analysis involved assembling the obtained sequences into transcripts and utilizing the Corset software for hierarchical clustering. The clustered sequences served as a reference for subsequent analyses, including quality control, annotation using seven databases (NR: NCBI non-redundant protein sequences; NT: NCBI nucleotide sequences; Kyoto Encyclopedia of Genes and Genomes) KEGG: http://www.genome.jp/kegg/ ; SwissProt: http://www.ebi.ac.uk/uniprot/ ; PFAM: http://pfam.sanger.ac.uk/ ; GO: http://www.geneontology.org ; KOG: http://www.ncbi.nlm.nih.gov/COG/ ), quantification, differential expression analysis, and functional enrichment. Section title: RT-qPCR validation Educational score: 4.104111194610596 Domain: biomedical Document type: Study Language: en The 2 -ΔΔCt was employed to compute the relative gene expression . In this investigation, we assessed the expression levels of four genes in diverse samples via RT-qPCR, examining gene alterations during infection. To facilitate the comparison of detected samples during the exponential phase of the RT-qPCR reaction, it is essential to first establish a specific fluorescence signal threshold. Typically, this threshold is determined based on the fluorescence signals from the initial 15 cycles of the PCR reaction, which serve as the fluorescence background signal. If a detected fluorescence signal surpasses this threshold, it is regarded as a true signal and can be utilized to define the threshold cycles (Ct) for the sample. There exists a linear inverse relationship between the Ct value of each template and the logarithm of the starting template’s copy number in the sample; thus, a higher starting copy number corresponds to a lower Ct value. Each specimen was analyzed in triplicate, and the Ct values were averaged. The primer sequences employed in this study are listed in Table 1 . Section title: Statistical analysis Educational score: 3.969266891479492 Domain: biomedical Document type: Study Language: en Most data analyses were conducted using software or built-in algorithms within the software packages. Feature enrichment tests were carried out using the GOseq package, while differential expression analyses were performed using the DESeq2 package. The criteria used for screening differentially expressed genes were | log 2 (Fold Change) | > 1, with a padj value of less than 0.05 ( P < 0.05). considered statistically significant. Section title: Statistical analysis Educational score: 3.9219439029693604 Domain: biomedical Document type: Study Language: en To assess the significance of differences between the means of two datasets, Independent Student’s t-tests were performed using SPSS version 22.0. This analysis included comparisons between RT-qPCR and RNA-seq data, with a p-value below 0.05 ( P < 0.05). indicating statistical significance, a p-value below 0.01 ( P < 0.01). indicating high significance, and a p-value above 0.05 ( P > 0.05) indicating no significance. Section title: Information on V. parahaemolyticus strain Educational score: 4.136902809143066 Domain: biomedical Document type: Study Language: en To analyze the virulence gene present in the strains, we conducted a gel electrophoresis (PCR) analysis. The cultured fresh colonies were identified using PCR, confirming the presence of several virulence genes: Gyr (629bp), TnaA (463bp), PyrC (533bp), RecA (773bp), and LuxR (679bp). Additionally, the virulence genes PirA (284bp) and PirB (392bp), which are associated with pancreatic necrosis, were also detected . The PCR products were sequenced by a sequencing company and subsequently compared with known virulence gene sequences of V. parahaemolyticus in the NCBI database. The results indicated that all identified virulence genes were pathogenic genes associated with V. parahaemolyticus. The antibacterial susceptibility test revealed that the macrolide antibiotic erythromycin exhibited no activity against the bacteria ( Table 2 ). Section title: Determination of LD50 and histopathological changes of hepatopancreas Educational score: 4.248628616333008 Domain: biomedical Document type: Study Language: en To analyze the pathogenicity of V. parahaemolyticus , we established an animal model to study the infection in Chinese mitten crabs. Subsequently, we assessed the median lethal dose (LD50) within a 24-hour period. The specific results at each time point are presented in Supplementary Table S1 . The LD50 of this pathogenic strain was calculated to be 1.78×10 6 CFU/mL using a formula calculation method . An animal infection model was established using the LD50 concentration. The changes in the hepatopancreas tissue of clinical animals were analyzed at various time points during the early, middle, and late stages of infection. The control group exhibited good condition and vigor, with crabs displaying active behavior, strong grip in their claw feet, and intact hepatopancreas with clear folds upon dissection. The gills appeared light cyan with neat filaments . After 6 hours of infection with V. parahaemolyticus , diseased crabs demonstrated significant abnormalities, weakened vitality, and reduced crawling ability. Dissection revealed a dark yellow appearance of the hepatopancreas, cloudy and blackened gills, and dilated stellate structures with contracted folds in the hepatopancreatic tissue . At 24 hours post-infection, crabs exhibited low vitality, moderate crawling ability, and a tendency to escape when disturbed. The hepatopancreas appeared significantly orange-yellow with slight erosion, and the gills turned black. Hepatopancreatic tissue displayed enlarged vacuoles, ruptured stellate structures, and continued expansion . By 72 hours after infection, the survival rate of diseased crabs was very low. They showed minimal activity, with no tendency to break free when handled. Dissection revealed necrosis in the hepatopancreas, eroded liver tissue with whitish coloration, and completely dark brown gills. The hepatopancreatic ductal structure exhibited increased folding contraction and extensive loss of nuclei . Section title: Tissue enzyme viability test Educational score: 4.149139404296875 Domain: biomedical Document type: Study Language: en Tissue enzyme activity serves as a critical indicator of the innate immunity in experimental animals. In this study, we utilized an animal infection model to collect hepatopancreas tissue from E. sinensis and measured various enzyme activity indicators, including ALT, AST, ACP, LZM, SOD, T-AOC, AKP, and PO. The results are presented as follows: changes in enzyme activities at various time points during infection were observed through the hepatopancreatic immune factor assay . Compared to the control group, the levels of ACP and T-AOC were significantly lower ( P < 0.05). However, there was a significant increase in the activity of SOD and lysozyme (LZM) within the initial 12 hours following infection ( P < 0.05). Subsequently, LZM activity decreased and was significantly lower than that of the control group ( P < 0.05). The results from the body fluid enzyme activity factor assay demonstrated significant activation of specific oxidative enzymes and lysozyme during the early phases of infection. Conversely, ALT and AST consistently exhibited significant activation throughout the entire infection cycle ( P < 0.05). Section title: Gene prediction and annotation Educational score: 4.230995178222656 Domain: biomedical Document type: Study Language: en During the process of E. sinensis resisting infection by Vibrio parahaemolyticus, the organism’s genes undergo differential transcription. Utilizing an animal infection model, we collected the hepatopancreas from experimental animals exhibiting various clinical symptoms during the infection. We analyzed the differential changes in its transcriptome through RNA-seq sequencing technology. The resulting data is presented as follows: A total of 205,181,269 raw data reads were obtained from the nine samples ( Table 3 ). Specifically, 69,600,138 raw reads were from the control group (Es_CG), 68,521,173 from the susceptible group (Es_SC), and 67,059,958 from the disease-resistant group (Es_AI). After screening, 20,215,931 high-quality Clean Reads were obtained, accounting for 98.53% of the total data. For transcript splicing, 68,583,106, 67,510,983, and 66,060,842 Clean Reads were obtained from the control, susceptible, and resistant groups, respectively. The raw data exhibited Q20 > 96.51%, Q30 > 91.56%, and GC content > 50.13%. Using de novo assembly with Trinity, a total of 232,924 genes were predicted. The shortest unigene was 301 bp, the longest 30,836 bp, with an average length of 991 bp. The N50 and N90 values were 1464 and 412, respectively ( Table 4 ). All sequencing raw data from this study have been deposited in the National Center for Biotechnology Information database under the accession number NCBI: PRJNA1130557. Section title: Statistical analysis of differentially expressed genes Educational score: 4.140050888061523 Domain: biomedical Document type: Study Language: en We utilized data analysis software based on the R programming language to conduct a statistical analysis of differentially transcribed genes across the three groups. For the differential expression of genes in E. sinensis infected with V. parahaemolyticus , we performed a statistical analysis. Initially, we calculated Pearson’s correlation coefficients using the FPKM values of all genes across the samples. The results, depicted in Figure 5 , demonstrated strong correlations between the samples (R^2 close to 0.9), indicating the data’s suitability for further analysis. Comparing ES_SC and ESCG, we identified 11,662 genes with differential expression, out of which 6,266 genes were up-regulated and 5,396 genes were down-regulated . Comparing ES_SC and ES_AI, A total of 13,515 genes exhibited differential expression, with 7,694 genes up-regulated and 5,821 genes down-regulated . Likewise, comparing ES_AI and ES-CG, we found 7,631 genes with differential expression, including 3,111 genes that were up-regulated and 4,520 genes that were down-regulated . To visualize the expression patterns of the differentially expressed genes, we generated a heatmap . This heatmap clusters genes with similar expression patterns together. Each square’s color indicates the expression level of the corresponding gene: redder colors denote higher expression levels, while greener colors denote lower expression levels. These analyses provide insights into the transcriptional responses of E. sinensis to V. parahaemolyticus infection, highlighting significant gene expression changes associated with susceptibility and resistance. Section title: Enrichment of differentially expressed genes Educational score: 4.3076491355896 Domain: biomedical Document type: Study Language: en The differential transcription of genes significantly influences the life and metabolic processes of experimental animals. In this study, we enrich and analyze the differentially expressed genes sequenced by RNA-seq using Gene Ontology (GO) enrichment and the KEGG database to elucidate how various hosts resist the invasion of pathogenic microorganisms through their own biological processes. The GO enrichment analysis revealed significant findings in the comparison between ES_AI and ES-CG groups. In terms of biological processes, genes demonstrated enrichment primarily in metabolic processes (149 genes, with 63 up-regulated and 86 down-regulated) and transmembrane transport (305 genes, comprising 152 up-regulated and 153 down-regulated). Cellular component analysis highlighted enrichment in the extracellular matrix (19 genes, with 13 up-regulated and 6 down-regulated). Molecular function analysis indicated significant enrichment in oxidoreductase activity (257 genes, 122 up-regulated and 135 down-regulated). In KEGG pathway analysis, up-regulated genes were notably enriched in pathways such as ribosome (54 genes) and protein processing in the endoplasmic reticulum (38 genes), while down-regulated genes were primarily associated with starch and sucrose metabolism (27 and 24 genes, respectively). These results provide valuable insights into the functional roles of differentially expressed genes in E. sinensis during infection with V. parahaemolyticus , highlighting key processes and pathways affected by the bacterial infection. Section title: Enrichment of differentially expressed genes Educational score: 4.247084617614746 Domain: biomedical Document type: Study Language: en The comparison between ES_SC and ES_AI demonstrated that the differential genes expressed in biological processes primarily exhibited enrichment in the metabolic process of cellular nitrogen compounds and the biosynthetic process as shown in Figures 7D–F . ES_SC had 972 down-regulated genes, while ES_AI had 794 down-regulated genes. Conversely, ES_SC showed 581 up-regulated genes compared to ES_AI’s 689 up-regulated genes. Furthermore, an enrichment of up-regulated genes was observed in the immune system process (20 genes), while 33 genes were down-regulated. Regarding cellular composition, genes showed significant enrichment in intracellular compartments and organelles. Specifically, ES_SC had 795 up-regulated genes compared to 619 in ES_AI. Conversely, ES_SC had 964 down-regulated genes, while ES_AI had 772 down-regulated genes. In terms of molecular functions, genes exhibited prominent enrichment in DNA binding and ion binding. The up-regulated genes were 311 in ES_SC and 433 in ES_AI, while the down-regulated genes were 456 in ES_SC and 433 in ES_AI. Additionally, the KEGG enrichment analysis revealed similar results, indicating that up-regulated genes mainly enriched the phagosome, pathways in cancer, and proteoglycans in cancer pathways, with 33, 37, and 33 enriched genes, respectively. On the other hand, down-regulated genes primarily enriched the cell cycle pathway and the ribosome pathway, with 44 and 64 enriched genes, respectively. Section title: Enrichment of differentially expressed genes Educational score: 4.312760353088379 Domain: biomedical Document type: Study Language: en When comparing ES_SC and ES-CG, the differential genes involved in biological processes are primarily enriched in the metabolic process of cellular nitrogen compounds and biosynthesis. There are 732 up-regulated genes and 611 down-regulated genes, along with 662 up-regulated genes and 556 down-regulated genes, respectively. Additionally, there are 22 up-regulated genes and 25 down-regulated genes enriched in the immune system process. In terms of cellular composition, genes are predominantly enriched in intracellular locations and organelles. Specifically, there are 838 up-regulated genes and 655 down-regulated genes enriched, while 648 up-regulated genes and 514 down-regulated genes are observed. Moving on to molecular functions, genes exhibit significant enrichment in DNA binding and ion binding. This enrichment involves 321 up-regulated genes and 507 down-regulated genes for DNA binding, and 302 up-regulated genes and 507 down-regulated genes for ion binding . Regarding the KEGG enrichment analysis, up-regulated genes are primarily enriched in pathways related to cancer, including pathways in cancer itself and Proteoglycans in cancer. The number of enriched genes is 46 and 49, respectively. On the other hand, down-regulated genes show enrichment in pathways related to starch and sucrose metabolism, as well as the lysosome pathway, with 31 and 40 enriched genes, respectively. Section title: Enrichment of differentially expressed genes Educational score: 4.259711742401123 Domain: biomedical Document type: Study Language: en The present investigation involved the screening of 691 genes from the immune-related functional classification provided by the KEGG database . In relation to immunity, a total of 342 differentially expressed genes were discovered. These selected genes exhibited enrichment in diverse KEGG pathways, with a particular emphasis on 22 signaling pathways associated with immune response . Amongst these pathways, notable considerations included platelet activation (37 genes), leukocyte transendothelial migration (29 genes), Fc gamma R-mediated phagocytosis (26 genes), chemokine signaling pathway (31 genes), toll-like receptor signaling pathway (20 genes), antigen processing and presentation (18 genes), T cell receptor signaling pathway (19 genes), NOD-like receptor signaling pathway (16 genes), B cell receptor signaling pathway (14 genes), Rap1 signaling pathway (38 genes), Fc epsilon RI signaling pathway (14 genes), TNF signaling pathway (16 genes), natural killer cell mediated cytotoxicity (12 genes), RIG-I-like receptor signaling pathway (11 genes), ErbB signaling pathway (15 genes), NF-kappa B signaling pathway (11 genes), Ras signaling pathway (28 genes), inflammatory mediator regulation of TRP channels (14 genes), GnRH signaling pathway (16 genes), cAMP signaling pathway (28 genes), PI3K-Akt signaling pathway (31 genes), and MAPK signaling pathway (27 genes). Several immune-related signaling pathways are graphically displayed in this study. The figure illustrates that infection with V. parahaemolyticus triggers the activation of specific genes within the KEGG signaling pathway in the samples analyzed. It is hypothesized that the activation of these genes plays a crucial role in initiating the respective KEGG signaling pathway . Section title: Transcriptome differential gene expression validation Educational score: 4.252246379852295 Domain: biomedical Document type: Study Language: en The validation results depicted in Supplementary Figure S2 demonstrate concordance between RNA sequences and qRT-PCR findings, supporting the accuracy and precision of RNA-Seq in this investigation. Temporal changes in the expression levels of 5 genes were analyzed at different time points (6, 12, 24, 48, and 72 hours) post-infection using qRT-PCR . The results revealed a significant increase in the expression of lysozyme-related genes at 6 hours after acute V. parahaemolyticus infection. Moreover, the redox-related SOD showed a consistent augmentation throughout the course of the infection, with the highest level of transcription attained at the 12-hour time point. Furthermore, the infection by V. parahaemolyticus led to an up-regulation of the integrin gene in the hepatopancreas tissue. The expression level of the integrin gene experienced a considerably significant rise at 6 hours post-infection ( P < 0.01), followed by a subsequent decline. However, at the 12-hour mark following infection, the integrin gene’s expression remained significantly up-regulated ( P < 0.01). Between the 24 and 72-hour periods after infection, there was a significant up-regulation of the integrin gene in crab hepatopancreas tissue ( P < 0.05), persisting at a sustained level below the normal range. Concurrently, the expression level of the hemocyanin HC gene in the hepatopancreas exhibited a notable rise ( P < 0.01), followed by a subsequent increase. The expression of HC peaked at 12 hours post-infection ( P < 0.01), after which it began to decline. Between 48 and 72 hours after infection, the HC gene expression returned to levels close to the baseline. Section title: Discussion Educational score: 4.1835455894470215 Domain: biomedical Document type: Study Language: en V. parahaemolyticus is a highly pathogenic bacterium known for causing significant mortality and economic losses in cultured E. sinensis . This study focused on V. parahaemolyticus -infected E. sinensis and involved constructing a transcriptome sequencing library from immune-related tissues in the hepatopancreas. This approach enabled the acquisition of gene sequences and functional information, substantially enriching the transcriptional expression and genetic information database of the crab. The expanded database will facilitate more comprehensive and systematic analyses of various aspects of crab biology, thereby aiding in management and disease control efforts. Additionally, we confirmed that the strains used in our study carry virulence genes such as Gyr , Tna , Pyr , Rec , Lux , and priA/B . Furthermore, these strains showed sensitivity to antibiotics including propoxur, norfloxacin, tetracycline, and piperacillin. Infection of laboratory animals with V. parahaemolyticus induced disturbances in genetic material and energy metabolism in the hepatopancreas of the Chinese mitten crab. Section title: Discussion Educational score: 4.371395111083984 Domain: biomedical Document type: Study Language: en The results of our study indicate that this particular strain of V. parahaemolyticus causes acute hepatopancreatic necrosis symptoms in E. sinensis . These symptoms resemble those observed in Chinese mitten crabs suffering from acute hepatopancreatic necrosis syndrome (AHPNS), a chronic disease affecting the hepatopancreas and energy consumption . Based on this similarity, we hypothesized that V. parahaemolyticus infection in Chinese mitten crabs could lead to increased depletion of hepatopancreatic energy, ultimately resulting in hepatopancreatic failure and necrosis as a pathological symptom. With prolonged infection duration, we observed significant changes in the color of the hepatopancreas in affected crabs, progressing from dark yellow to white. Additionally, the tissue structure exhibited severe necrosis. Pathological tests revealed deformations in the stellate lumen structure of hepatic tubules in the hepatopancreas of infected crabs, alongside the appearance of vacuoles in hepatocytes, followed by gradual lysis. The hepatopancreas serves as a multifunctional organ integrating metabolic and immune functions, making it the primary target tissue for V. parahaemolyticus AHPND infection in crustaceans . V. parahaemolyticus infection directly caused acute hepatopancreatic injury, suggesting it could contribute to acute hepatopancreatic necrosis in Chinese mitten crabs. Section title: Discussion Educational score: 4.2487993240356445 Domain: biomedical Document type: Study Language: en As typical hydrolases in the innate immune system of crustaceans, alkaline phosphatase (ACP), acid phosphatase (AKP), and lysozyme (LZM) primarily function to directly eliminate extracellular invaders. Therefore, they are considered sensitive biomarkers in the context of environmental stress and microbial infections . Our study observed an increase in the activity of these enzymes during the early stage of infection, followed by inhibition in the late stage. Based on the analysis of enzyme activity measurement results and pathological sections, we speculate that damage to the hepatopancreas tissue may be one of the reasons for the contribute to the observed decrease in enzyme activity. Further experimentation involving pollutant exposure demonstrated a correlation between decreased enzyme activity in fish and reduced activity of liver and blood cells , indirectly supporting our initial speculation. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are both aminotransferases that belong to the same class and are involved in protein metabolism within the body. These enzymes are primarily located in cellular mitochondria. Changes in their activity can indicate alterations in metabolism or tissue damage . In our experiments, we observed significant pathological damage to the hepatopancreas of infected crabs. Additionally, the activities of ALT and AST changed over time during infection, suggesting that the protective mechanism of the crab’s hepatopancreas was impacted by V. parahaemolyticus infection. This finding supports the idea that the increased mortality rate observed in Chinese mitten crabs after V. parahaemolyticus infection is mainly due to hepatopancreatic damage . Section title: Discussion Educational score: 4.462696075439453 Domain: biomedical Document type: Study Language: en Oxygen free radicals in aquatic animals exist in a dynamic equilibrium, which can be disrupted by bacterial invasion or other stressors, leading to the generation of large amounts of free radicals. The organism has the ability to activate its antioxidant defense system to eliminate reactive oxygen species and prevent oxidative damage. However, excessive oxidative stress can contribute to disease development. SOD is an enzyme that plays a crucial role in the antioxidant system of organisms and serves as an important component of innate immunity in crustaceans . In vitro bacterial agglutination assays of manganese SOD (MnSOD) have demonstrated its ability to agglutinate a wide range of both Gram-negative and Gram-positive bacteria . During infections caused by V. parahaemolyticus or Staphylococcus aureus , total SOD activity and mRNA levels of MnSOD were significantly increased, indicating the involvement of MnSOD in antimicrobial immunity through regulation of the cellular redox state . It is hypothesized that bacterial infection may induce alterations in the levels of oxygen free radicals in crabs during the initial phases of infection. SOD and glutathione, as primary cellular antioxidants, work to eliminate excess superoxide ions in tissues. Section title: Discussion Educational score: 4.530947208404541 Domain: biomedical Document type: Study Language: en During RNA-seq, analysis of differentially expressed genes using enrichment techniques revealed a significant overrepresentation of immune genes in multiple signaling pathways. These pathways include the Toll-like receptor (TLR), mitogen-activated protein kinase (MAPK), immunodeficiency (IMD), and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways. Innate immunity, a shared characteristic between invertebrates and mammals, is recognized as the primary defense against microbial pathogens . In invertebrates, activation of the Toll and IMD pathways occurs upon invasion by pathogenic molecules, triggering a host defense response. Crustaceans also possess crucial innate immune pathways that play pivotal roles in combating microbial infections . When external pathogenic microorganisms breach the physical barriers of crabs, immune signaling pathways such as Toll, IMD, and JAK/STAT are activated. Regulatory effects of innate immunity, including activation of complement and coagulation cascades, phagocytosis, pro-inflammatory signaling cascades, and apoptosis, are essential components of the host immune response. These processes involve multiple elements such as antimicrobial peptides (AMPs), complement-like proteins, and blood cells . In invertebrates, various self-defense mechanisms contribute to the innate immune response. In crustaceans, these mechanisms primarily involve AMP-mediated bacterial killing, generation of reactive oxygen species through the prophenoloxidase-activated system, phagocytosis of microorganisms by hemocytes, and the melanin deposition cascade for microorganism entrapment . Significant differences were observed in the transcription of genes encoding SOD, CDSP, integrin, LZM, HC and their derivatives. Our study provides valuable insights into the activation of signaling pathways during V. parahaemolyticus infection in Chinese mitten crabs. Section title: Conclusion Educational score: 4.189898490905762 Domain: biomedical Document type: Study Language: en Following infection with V. parahaemolyticus , Chinese mitten crabs primarily exhibit hepatopancreatic necrosis as the predominant clinical manifestation. Transcriptomic analysis of the hepatopancreas from infected crabs revealed successful annotation of a substantial number of genes across seven major databases. A total of 342 genes associated with immune response displayed differential expression, notable enrichment in both the Toll and MAPK pathways. Our findings indicate that activation of the immune defense mechanism in crabs due to by V. parahaemolyticus significantly enhances hepatopancreatic function, as well as the activities of antioxidant and immune-related enzymes within the hepatopancreas. | Study | biomedical | en | 0.999995 |
PMC11695294 | Section title: Introduction Educational score: 1.038457989692688 Domain: other Document type: Other Language: en Padel stands out as one of the emerging sports, experiencing unprecedented growth in recent years, both in terms of participants and the number of facilities. Its popularity is so high that the number of padel players surpasses that of tennis players in countries like Spain, Sweden, and Portugal. Notably, there has been an increase in participation, international expansion of padel infrastructure, and its growing economic importance ( 1 ). Section title: Introduction Educational score: 1.1149312257766724 Domain: other Document type: Other Language: en The consolidation of padel as a sport of growing popularity and accessibility in various social contexts can be attributed to multiple factors that favour its practice and dissemination: easy to learn, suitable for different ages, gender, abilities or physical condition, or economic access to practice facilitating its democratization. In addition, it encourages the friendship or peer group factor, satisfaction or fun ( 2 ), social engagement and interaction among participants, promoting values of cooperation and mutual respect. The combination of these factors contributes significantly to the rise and consolidation of padel in today's sporting landscape ( 3 ). Section title: Introduction Educational score: 1.0588619709014893 Domain: other Document type: Other Language: en The increase in the number of people interested in padel comes both from the players themselves and from those attending sporting events related to the sport ( 4 ). In Spain, López ( 5 ) indicated that it is the sport with the greatest development in the last 23 years with a growth of 1,947.41%, more specifically 20% in the last 10 years according Courel-Ibáñez et al. ( 6 ), being also the country with the highest number of padel clubs between 2019 and 2021 with a growth of 11%, experiencing the highest Google search for the word padel ( 1 ). This situation is transferred to the evolution of the number of licences, whose evolution since 2012 has shown a permanent growth experiencing its hatching in the post-pandemic period thanks to the fact that its practice takes place, mostly, in outdoor facilities. It has gone from 75 thousand licenses in 2020 to over 90 thousand in 2021 ( 7 ). In terms of padel facilities, Spain is the country with the highest number of courts in the world (15,300), with 279 million euros spent on the construction of courts in the last two years ( 1 ). All these data indicate that padel is one of the most emerging and fastest-growing sports of the 21st century ( 8 ). Section title: Introduction Educational score: 1.1336184740066528 Domain: other Document type: Other Language: en The evolution experienced in the practice of padel and its economic impact are indicative of the importance of adapting and continuously improving the quality of the services offered. In fact, this impact of padel has focused the attention of the scientific community with recent studies aimed at analysing variables that affect the game and/or players,. however there is a significant lack in the academic literature of studies oriented to the evaluation of the perception of padel users or players on quality, despite its extensive analysis in a variety of sports organisations as a result of the importance of its study as a starting point to achieve satisfaction and future behavioural intention. Section title: Introduction Educational score: 1.1793204545974731 Domain: other Document type: Other Language: en Service quality starts from the satisfaction of expectations, i.e., from the comparison of expectations with the service they perceive they have received ( 9 , 10 ), and has been a widely analysed construct in the sport industry and specifically in sport services [e.g., ( 11 – 14 )]. In the context of padel and in the face of the latent emerging demand for associated sport services, it is not an easy task to provide satisfactory and high quality services to all customers ( 15 ). In this way, knowing which attributes padel users consider most relevant is decisive for an adequate quality management within the service provision process. In this sense, the validation of quality assessment tools specific to padel becomes a critical resource for measuring and improving the user experience ( 16 ). Section title: Introduction Educational score: 3.1523334980010986 Domain: other Document type: Study Language: en However, there has been an increase in scientific research whose main focus is on areas such as performance analysis, psychology and physiology, while sport management occupies a less prominent place in the literature as reflected in the systematic review by Sánchez-Alcaraz et al. ( 17 ). Specifically, research oriented towards sport management aims to analyse the evolution of federal licences ( 3 , 8 ), an analysis of the World Padel Tour in terms of expenditure per attendee ( 18 ) and the cost-benefit of its organisation ( 19 ), the standardisation of facilities ( 20 ), the viability of padel centres for the development of business plans ( 21 ), and a study on user satisfaction ( 22 ), which is the most relevant to the present research, involving two padel clubs and a sample of 36 participants. Therefore, we consider that there is a notable lack of research aimed at assessing the perception of padel users, and in parallel this situation suggests a potential opportunity for the validation of instruments that allow the assessment of constructs as relevant as perceived quality in this specific context. In this way, achieving high levels of quality in service provision is considered one of the indispensable requirements when it comes to obtaining adequate competitiveness and viability in organisations, and for because of this it is necessary to pay attention to all the components surrounding the service in order to achieve the greatest possible homogeneity in them ( 23 ). Section title: Introduction Educational score: 1.7624481916427612 Domain: other Document type: Other Language: en In this context, perceived quality in sport services emerges as a fundamental concept within the interaction that takes place between organisations and their users, being fundamental for the success of organisations and the loyalty of their customers ( 24 ), as well as for the increase of competitiveness ( 25 ). Within this relationship, the theory of perceived quality suggests that consumers evaluate the quality of a service based on the discrepancy between their prior expectations (or expected service) and their actual perception of the service received (the way it has been performed) ( 9 , 26 , 27 ). Based on this, the two main models are founded on a technical or result dimension and a functional or process dimension ( 9 ). On the other hand, series of attributes or dimensions such as tangible elements, reliability, responsiveness, safety and empathy ( 27 ), give rise in this second case to one of the most widely used tools (SERVQUAL). This tool has been reconfigured, tested and modified to be useful for measuring customer perceptions in a variety of industries ( 28 ) despite the various criticisms received for its lack of specificity or universal applicability ( 29 – 32 ) and even for psychometric aspects ( 33 ). This is why the best system is one that is generated or adapted to each organisation according to its characteristics and needs ( 23 ). Section title: Introduction Educational score: 1.15037202835083 Domain: other Document type: Other Language: en Perceived quality is currently still the subject of research in various sectors. Specifically in the sport industry and especially in sport services, understanding the relationship of service quality and customer experience to be crucial to improve customer loyalty and in turn build long-term relationships ( 34 ), with previous studies revealing that service quality improves both customer satisfaction and loyalty ( 13 , 35 , 36 ), as does experiential quality ( 37 ). In addition to the numerous studies using the SERVQUAL scale by Parasuraman et al. ( 27 ), other tools available for the evaluation of sport services are the QUESC scale ( 38 ), the SQFS scale ( 39 ), the SQAS scale ( 40 ) and its reduced version SQAS-19 ( 28 ), the SQS-FC scale ( 41 ), the QSCSEF scale ( 42 ), the CALIDFIT scale ( 43 ) and the CECASDEP scale and its reduced version ( 12 , 44 ), among others. There are also validated tools for other contexts, such as the SSQRS scale for recreational sports ( 45 ), the EVENTQUAL scales ( 46 ) or Eventserv ( 47 ) and its reduced version Eventserv-Short ( 48 ) for spectators of sporting events. Participants also receive attention in different contexts such as fantasy sports websites ( 49 ), running races ( 50 ), duathlon ( 51 ), or open water swimming ( 52 ). Section title: Introduction Educational score: 3.024620771408081 Domain: other Document type: Study Language: en In the specific context of padel, the study by Aparicio-Sarmiento et al. ( 22 ) used the EPOD scale ( 53 ) keeping the scale intact with 28 items distributed in 4 dimensions (sports technicians, material resources, activities, and image of the organisation), a tool that was subsequently validated in a sample of athletes participating in physical activities in a multi-sport centre using a 29-item version ( 54 ). In the study by Aparicio-Sarmiento et al. ( 22 ), although they reported adequate internal consistency ( α = 0.92), this is a value corresponding to the global scale and no information is provided on the psychometric properties of the tool applied to this specific context (exploratory and confirmatory factor analysis). Therefore, we consider it necessary to explore new options for the assessment of perceived quality in this context since, as stated by Haensel and Hoffmann ( 55 ), the dimensions of service quality can be significantly different depending on the type of business. Section title: Introduction Educational score: 3.712114095687866 Domain: biomedical Document type: Study Language: en Based on different reviews in the scientific literature ( 40 , 56 – 59 ) and extending it, Table 1 presents the main dimensions of a total of 34 studies that use different tools or adaptations for the evaluation of perceived quality in different sport services. The use of the dimensions “staff” and “programmes” are the most frequent in the literature, forming part of 33 and 25 scales, respectively. In the case of “facilities”, a dimension present in 28 scales, there are different terminologies to refer to the infrastructure that enables sport practice, although certain scales use what we could call a second level when referring to “sport spaces”, understanding these as the specific enclosures provided with the necessary means that allow practice (including here other uses such as learning or training and competition). In this sense, the scales CECASDEP, CECASDEP-R, SQAS and the adaptation of SQAS establish a differentiation between the physical installation and the specific facilities or spaces for carrying out an activity or exercise (e.g., gymnasium, swimming pool, or in the case of the present research, padel court), while in the case of the EPOD2 scale, the 3 items of the so-called “spaces” dimension refer to changing rooms (2 items) and cleanliness (1 item). Material” or “equipment” is another dimension frequently used in different scales, using some of these terms to refer to the elements that allow the adequate development of the contents during sport practice. Finally, other dimensions used in some scales have been included, such as tangible elements, cleanliness, changing rooms, user care, or information and communication, among others. Section title: Introduction Educational score: 2.0289947986602783 Domain: other Document type: Study Language: en Therefore, the objective of this work is focused on adapting a perceived quality assessment tool to the padel context, analyzing the psychometric properties necessary for its validation using a sample of users of sports facilities and services of padel, thus responding to the gap existing in the academic literature in a specific context of great relevance today. Section title: Participants Educational score: 2.717261552810669 Domain: biomedical Document type: Study Language: en The sample consisted of a total of 402 padel users (298 men and 104 women), all from the Autonomous Community of Andalusia. In relation to the characteristics of the sample, the age range of the participants was between 18 and 68 years old, with the highest percentages concentrated in the 19 and 21 age group (9.2%, 9.0% and 12.2%, respectively), 63.7% of the participants indicated that they were not members of a padel club, 73.4% practised padel in a private facility, while in terms of occupation, percentages close to 50% were obtained for both students (48%) and workers (52%). In relation to proximity, 77.1% indicated that it took them less than 15 min to get to the sports facility, and in relation to the practice profile, 61.4% played padel between 1 and 2 times a week, with the majority using between 1 and 2 h as playing time per day (89.6%) ( Table 2 ). Section title: Instrument Educational score: 1.8580313920974731 Domain: other Document type: Study Language: en The tool used in this study is adapted from a previous research work developed in the context of sports services ( 12 ), and has been adapted by research carried out in Mexico ( 85 ), Chile ( 86 ), Ecuador and Colombia ( 87 ) in the context of sports services, obtaining adequate psychometric properties. Section title: Instrument Educational score: 2.028315544128418 Domain: other Document type: Study Language: en For the adaptation to the context of padel, a committee of 4 experts of the Degree in Physical Activity and Sport Sciences from different universities was created, of which 2 were specialists in racket sports and sports management, 1 specialist in sports management and owner of a padel club, and 1 specialist in methodology, all with 15 years of professional experience. The Delphi methodology ( 88 , 89 ) was used to configure the committee of experts, guaranteeing anonymity at all times between the participating members of the committee. The process had 2 rounds of review, analyzing the relevance of the items and making the necessary. Thus, the name of 3 dimensions of the original tool was modified, specifically padel courts instead of sports spaces, padel activity programme instead of activity programme, and padel technicians instead of teacher-monitor. On the other hand, 1 item of the padel courts dimension was eliminated as it lacked possible adaptation (“the acoustics of the sports spaces are good”), and the content of some items was slightly adapted to be specifically oriented to the context of padel (e.g., “the external dimensions of the courts where I play are adequate” instead of “the dimensions of the space where the activity takes place are adequate”, or “the courts offer me safety” instead of “the sports space offers safety”). Section title: Instrument Educational score: 1.843788743019104 Domain: other Document type: Study Language: en Thus, the version used for the present research was a model composed of forty-eight items maintaining the five-point response format (1 = “I do not agree at all” to 5 = “I strongly agree”) and the five original dimensions: sports facility (ID, 10 items), padel courts (PP, 9 items), changing rooms (V, 12 items), padel activities programme (PAP, 9 items), and padel technicians (TP, 8 items). A specific block was included with the following socio-demographic questions: age, gender, level of studies, professional situation, type of club where padel is played, time to get to the padel facility, years of practice, days of practice per week, hours of practice per day and amount of money spent per month to practice padel. Section title: Procedure Educational score: 1.6777900457382202 Domain: other Document type: Study Language: en The Andalusian Padel Federation was contacted and agreed to participate in the research by providing the link to access the tool through their website. Data were collected over a 12-month period, specifically between 23 April 2022 and 23 April 2023. The questionnaire was administered in an online format created with Google forms, with all questions set to mandatory to eliminate non-response bias. The wording requested voluntary participation and guaranteed the anonymity of the responses, followed by the obligation to mark informed consent in order to continue with the process. The questionnaire took approximately 8–10 min to complete. Section title: Data analysis Educational score: 4.140830993652344 Domain: biomedical Document type: Study Language: en Descriptive statistics (means and standard deviation) and the normality of the data were calculated using univariate skewness and kurtosis values. To check the internal structure of the questionnaire adapted to the context of padel, an exploratory factor analysis (EFA), principal component analysis and oblimin oblique rotation were carried out, checking the relevance through Bartlett's test of sphericity and the Kaiser-Meyer-Olkin test (KMO), in addition to the percentage of variance explained. Measures to check the quality of the results were communalities [≥0.50; ( 90 )] and factor weights [≥0.50; ( 91 )]. The internal consistency of the dimensions was assessed using Cronbach's Alpha [α≥; ( 92 )]. Several measures were used to assess model fit quality in confirmatory factor analysis (CFA): (1) χ 2 and its differences of degrees of freedom [ χ 2 /df ≤ 3; ( 93 )], the comparative fit index (CFI), the incremental fit index (IFI), the Tucker–Lewis index (TLI), parsimony comparative fit index (PCFI) and root mean square error of approximation (RMSEA). According to Geiser ( 94 ), a model with a good fit to the data is characterised by CFI, IFI and TLI values above 0.90 and RMSEA values of 0.08 or lower, while values ≥0.60 are adequate for the PCFI indicator ( 63 ). Additionally, composite reliability [CR > 0.70; ( 95 )], average variance extracted [AVE > 0.50; ( 95 )], convergent and discriminant validity were calculated. Section title: Results Educational score: 3.975615978240967 Domain: biomedical Document type: Study Language: en Normality test results showed adequate skewness and kurtosis values for all variables, falling within the conventional criteria for normality [±3; ( 96 )]. The mean value of the dimensions showed a very low difference (range of 0.30), namely between 3.96 for the dimensions sports facility (SD = 0.34) and changing room (SD = 0.96) and 4.26 for the dimension padel technicians (SD = 0.92). Within this first phase of analysis, the internal consistency of the dimensions was checked by means of Cronbach's Alpha indicator, obtaining in all cases values above the recommended 0.90. Section title: Results Educational score: 4.155145645141602 Domain: biomedical Document type: Study Language: en The relevance of the EFA provided satisfactory results for both Bartlett's test of specificity [ χ 2 = 16103.81; p < 0.001] and the KMO test (0.956), thus showing an adequate factor structure for the different dimensions that explains 69.70% of the variance. The EFA results showed communalities above 50% for all items except for items ID4 (0.45) and ID5 (0.47), and factor loadings for the items above 0.50. The factor structure of the EFA was assessed by means of a CFA in order to test the model fit, using a maximum likelihood estimation method to determine how the items represented the different constructs. The results obtained showed a satisfactory fit according to the different indices considered, with a χ 2 /df value below 3 (2.17), CFI (0.92), IFI (0.92) and TLI (0.91) values above the minimum cut-off point, the PCFI indicator (0.86) above 0.60 and the RMSEA index [0.054 (LO = 0.051; HI = 0.057)] below the 0.08 cut-off point. In the case of the factor loadings between observable variables, they showed high values, above 0.60 in all cases, without the need to eliminate any item, as good results were obtained in all cases in all the analyses carried out to check the psychometric properties ( Table 3 ). Section title: Results Educational score: 4.055791854858398 Domain: biomedical Document type: Study Language: en Additionally, complementary measures were analysed to test the reliability and validity of the tool. For the first case, the composite reliability (CR) obtained values higher than 0.70 in all the constructs, its being able to affirm that the items of the manifest variables really measure each of the underlying variables ( 97 ). For the second case, the average variance extracted (AVE) was used, which represents the proportion of variance of the construct that can be explained by its indicators, obtaining values higher than the minimum value 0.50 assuming convergent validity, and convergent validity was also analysed using the Fornell-Larcker ( 98 ) criterion, where squared correlations were obtained between constructs lower than their respective AVE values ( Table 4 ). Section title: Discussion Educational score: 1.90066397190094 Domain: other Document type: Study Language: en The relevance of padel as an emerging sport has attracted attention in the scientific literature in recent years, with several studies focusing on aspects related to the players (performance, psychology, or physiology) as well as certain variables associated with sports management, but the quality of the service has not been adequately addressed. The aim of this study was to adapt and validate a tool to assess the quality perceived by users of padel facilities and services, using a sample of users of this type of facilities and services from the Autonomous Community of Andalusia, a region located in the south of Spain. Section title: Discussion Educational score: 3.1394894123077393 Domain: other Document type: Study Language: en Aparicio-Sarmiento et al. ( 22 ) focused their study on the analysis of the satisfaction of padel users in a sample of 36 participants, using the EPOD tool ( 53 ), originally designed for the evaluation of user satisfaction in sports organisations. However, the items used in the study have a shorter wording than the original version without describing the adaptation process used, without providing information on the psychometric properties, as well as using the construct satisfaction and not perceived quality. Perceived quality implies according to Bitner and Hubbert ( 99 ) a consumer's impression of the relative superiority or inferiority of an organisation and its services, whereas satisfaction implies a post-consumer ( 100 ) or post-purchase ( 101 ) response or evaluation, hence quality is considered an antecedent of satisfaction ( 62 ) and satisfaction an antecedent of behavioural intention ( 102 ). This is in line with Baena-Arroyo et al.'s ( 35 ) assertion that perceived quality is a first step to customer loyalty, while other variables such as satisfaction are determinants of consumer behaviour ( 87 ). Section title: Discussion Educational score: 1.6157821416854858 Domain: other Document type: Study Language: en Perceived quality is one of the variables that have received most attention in the sport industry. In the present study, with the intention of filling a gap in the literature, a review of tools designed for the evaluation of perceived quality in different contexts within sport services was carried out, considering the use of the CECASDEP tool for adaptation to the context of padel. Its internal structure integrates 3 of the most commonly used dimensions in the literature (see Table 1 ), namely personnel (referring to the human resources and workers of the specific service), programmes (referring to the contents, services and activities of padel), and facilities (referring to the global installation that integrates the different sports spaces necessary for the development of the sport activity). Section title: Discussion Educational score: 2.838111162185669 Domain: other Document type: Study Language: en These 3 dimensions include a specific dimension for the changing room and a dimension for the playing space itself (padel courts). In the case of the changing room, it is a space necessary at a functional level (e.g., change of clothes, hygiene, safety through lockers), and at the level of user experience (pleasant atmosphere, cleanliness, additional or complementary equipment, etc.), including in both cases adaptability and accessibility for people with functional diversity. However, this dimension is only used in the SQAS tool ( 40 ) and in the adaptation made by Yu et al. ( 80 ), although there are some tools that address this space, although not in such a specific way. This is the case of the QUESC scale ( 38 ) which includes the dimension “ambiance” with 7 items, 3 of them referring to changing rooms (comfortable temperature, warm changing room, and cleanliness); the HATSQ scale ( 70 ) includes the dimensions “ambience and accessibility” where 2 items refer to changing rooms (changing rooms and hygiene in the shower area); the NEPTUNO scale ( 73 ) included a specific dimension for cleanliness where they dedicated 1 item to ask about the cleanliness of the toilets; the EPOD scale used by Aparicio-Sarmiento et al. ( 22 ) for the satisfaction of padel clubs refers to the changing room using only 1 item to assess cleanliness. As for the specific dimension on padel courts, no tools have been found that address this specific sports space, so the present study adapted the original dimension called “sports spaces” as padel courts are considered a determining factor in the perception of service quality, this criterion contributing to a better positioning in a competitive market. The adaptation process led to the elimination of 1 item that lacked the possibility of adaptation due to lack of application to the specific context (“the acoustics of the sports spaces are good”), and the remaining items addressed aspects related to dimensions, lighting, playing surface, net, maintenance, cleanliness, orientation and safety. Section title: Discussion Educational score: 3.255011558532715 Domain: other Document type: Study Language: en The results obtained in this study show adequate psychometric properties of the CECASDEP tool adapted to the context of padel. Given the importance of the analysis of perceived quality in the sports sector, it is crucial to have tools that comply with a rigorous methodology and that allow a valid and reliable assessment to be carried out. In this sense, the QPadel tool has an internal structure of 5 dimensions and 48 items that allow the evaluation of the perceived quality of users of padel facilities and services, and also represents a novel contribution to the literature as it is a validated tool for a context in which there is a significant gap. Additionally, it has a great potential for adaptability to different contexts related to racket and padel sports, and consequently, to different practical realities by providing an important aid to managers of padel facilities for the analysis of the perceived quality of their customers and users, facilitating the establishment of strategies to generate a service that allows a better adaptation to the needs and interests. Section title: Practical implications Educational score: 1.9055331945419312 Domain: other Document type: Other Language: en The QPadel tool, adapted and validated for the present study, makes an important contribution to the literature by filling an existing gap and has a broad applicability by providing the possibility to assess the quality perceived by users. Therefore, and considering the relevance of providing a superior service for the survival of a service organisation ( 103 ), the assessment of perceived quality will facilitate decision-making and the use of available resources for the improvement of quality, and consequently, satisfaction and loyalty. Section title: Practical implications Educational score: 1.5640240907669067 Domain: other Document type: Other Language: en The results obtained confirm that the dimensions used are relevant in the context of padel, obtaining adequate psychometric properties in the different analyses developed. This is especially remarkable in a context that has experienced a high boom and growth, as it provides organisations with a reliable and valid tool. Section title: Practical implications Educational score: 1.2967716455459595 Domain: other Document type: Other Language: en Focusing on managers, the results of this study have important implications considering the relevance of the information provided by each of the dimensions, impacting positively on the development of competitive strategies by allowing the identification of areas for improvement in aspects related to these dimensions, such as including improvements in infrastructure, providing specific training for staff, or the creation of programmes of activities and/or events tailored to the needs of users. Therefore, we want to encourage sport managers of padel-related facilities to work with a validated tool that facilitates the establishment of quality standards within the padel industry. It can also serve as a starting point for a benchmarking process that promotes healthy competition and an overall increase in the levels of quality offered to users. Section title: Practical implications Educational score: 1.244403600692749 Domain: other Document type: Other Language: en We believe that the implementation of the findings of this study can help to achieve a significant improvement in the management and operation of padel sport facilities, increasing user satisfaction and loyalty and strengthening the competitive position of organisations. Section title: Limitations and future directions Educational score: 2.5457053184509277 Domain: other Document type: Study Language: en Future studies need to be aware of the limitations found in the present research. The volume of users in padel clubs is not comparable to that of other sporting activities and the frequency of use is also lower, so the sample collection is not an easy task and could not be carried out in a short space of time. Despite the support of the Andalusian Padel Federation, which facilitated the distribution of the tool throughout the Autonomous Community of Andalusia, we were not able to make a specific selection of the participating padel clubs and this provided an imbalance between public and private clubs (types of facilities). Another imbalance was obtained in the gender variable, where the sample of women was much lower than that of men and prevented us from going deeper into specific perceptions and analysing the possible existence of differences. In relation to the frequency of use (weekly practice), we consider it necessary to increase the participants of users who practice padel between 3 and 4 times a week by adding a specific question that allows us to identify whether they practice in the same sports facility or not, so that we can identify whether there is any reason other than proximity (convenience of service) that is associated with the perceived quality, and also, consider the variables age and level of play to check for possible discrepancies in perceived quality, all aimed at better management of both the facilities and the services offered. Section title: Discussion Educational score: 3.2580959796905518 Domain: biomedical Document type: Study Language: en The results obtained in this study show adequate psychometric properties of the QPadel assessment tool, which comes from the adaptation of another perceived quality evaluation tool after an indepth review of the literature. Specifically, QPadel responds to the consideration of the three dimensions most commonly used in the literature (facilities, program and staff). These dimensions include items that encompass user attention, location and external equipment (facilities), characteristics and expectations (programme), and, finally, content and interaction (staff). In addition, this tool dedicates a specific dimension to the sports space where the activity takes place, including items on comfort and functionality, and last of all, in the case of the changing room dimension, it includes items on environmental elements and comfort. Section title: Discussion Educational score: 1.4391614198684692 Domain: other Document type: Other Language: en Given the importance of the analysis of perceived quality in the sports sector, it is crucial to have tools that comply with a rigorous methodology and that allow a valid and reliable assessment to be carried out. In this sense, the QPadel tool has an internal structure of 5 dimensions and 48 items that allow the evaluation of the perceived quality of users of padel facilities and services, and also represents a novel contribution to the literature as it is a validated tool for a context in which there is a significant gap. Additionally, it provides important help to managers of padel facilities for the analysis of the perceived quality of their customers and users, facilitating the establishment of strategies to generate a service that allows a better adaptation to the needs and interests. | Other | other | en | 0.999998 |
PMC11695295 | Section title: Introduction Educational score: 4.0028204917907715 Domain: biomedical Document type: Review Language: en Child development refers advancement of the child in all areas of human functioning: social and emotional, cognitive, communication and movement ( 1 ). The first 1,000 days (from conception to 2 years) is a critical period for brain development ( 2 ). The majority of organ development, including the nervous system occurs before 5 years of age. About 90% of the size of the child’s brain grows in the first 5 years of a child’s life ( 3 ). Thus, early childhood is the most vulnerable period for the occurrence of developmental delays ( 4 ). Delayed child development refers to a child who does not achieve the developmental skills expected of his or her age compared to other children of the same age ( 5 , 6 ). The American Academy of Pediatrics recommends screening children for developmental delay at 9, 18, and 30 months of age with standardized tools ( 7 ). The standardized tools that are widely used to screen children for developmental delay in low-income countries, including Ethiopia are the Denver Developmental Screening Test II ( 8 ), the third edition of Ages, and Stages Questionnaires (ASQ-3) ( 9 ), and the Bayley Scale of Infant Development III ( 10 ). Section title: Introduction Educational score: 2.8536057472229004 Domain: biomedical Document type: Other Language: en Delayed child development is a global, regional, and national problem. Globally, 8.4% of children younger than 5 years had developmental disabilities in 2016. Almost all (95%) of these children lived in low-income and middle-income countries ( 11 ). According to global statistics, about 5–16% of children around the world have developmental disorders. Approximately 30–50% of these disorders are not identified until school age and therefore cannot be treated ( 12 ). More than 250 million, approximately 43% of children living in low-income and middle-income countries have some form of developmental delay, where the burden is highest in Sub-Saharan Africa accounting for 66% in 2010 ( 13 – 15 ). Section title: Introduction Educational score: 2.092503070831299 Domain: biomedical Document type: Other Language: en Developmental delays affect a child’s learning ability and emotional development which negatively impact income or productivity, and their future life. Poor growth and undernutrition are also associated with delayed mental development which leads to poor school performance and reduced intellectual achievement ( 16 ). Globally, the average loss of income or productivity due to poor child development is estimated at 19.8% ( 15 ). Section title: Introduction Educational score: 2.46453595161438 Domain: biomedical Document type: Other Language: en Many factors, including poor health of pregnant mothers, birth complications, very low birth weight, infections, genetic characteristics, exposure to toxins, trauma, and perhaps low socioeconomic status ( 17 , 18 ), an increase in the use of electronic devices, even among young children ( 19 ) and internal migration ( 20 ) are related to increased risk of developmental delay. Section title: Introduction Educational score: 2.112907648086548 Domain: biomedical Document type: Other Language: en In Ethiopia, the burden of child developmental delay cannot be explained solely by poor health or undernutrition of young children, further aggravated by the lack of sensitive and responsive child care, feeding, stimulation, and safety or security, all of which result in an estimated 59% of children under 5 years of age being at the risk of suboptimal development in the country ( 21 ). Section title: Introduction Educational score: 1.7800592184066772 Domain: biomedical Document type: Other Language: en In Ethiopia, the Early Childhood Development (ECD) initiative strategic plan is being implemented under the child health program aiming that all children grow and thrive in a secure, safe, and nurturing environment ( 22 ). In addition, system and policy changes can influence access to health care for the prevention and treatment of illness, malnutrition, early marriage, and maternal education, all of which may affect child development ( 23 ). Section title: Introduction Educational score: 2.253795862197876 Domain: biomedical Document type: Study Language: en Though the above strategies are being implemented, the prevalence of developmental delay among children in Ethiopia remains a public health problem. Therefore we aimed to assess the prevalence of developmental delay and its associated factors among children aged 6–59 months in Dembecha district, Northwest Ethiopia. Section title: Study area Educational score: 2.1811773777008057 Domain: biomedical Document type: Study Language: en The study was conducted in Dembecha district which is one of the 15 districts in the West Gojjam Zone, Amhara Regional State in Northwest Ethiopia. It is located 350 km from Addis Ababa in the Northwest direction, 203 km from Bahir Dar in the South direction, and 38 km from Finote Selam in the East direction. There were 6 health centers and 27 health posts (the lowest administrative primary health care unit that provides basic health services to the community in Ethiopia), which provide basic health services to the people of the district by focusing on maternal and child health. The climatic condition was 83% woyna dega 11% dega (vegetated cool zone with an altitude of over 2,600 m above sea level) and 6% Qola (hot zone with an altitude of below 1,500 m above sea level). The population of Dembecha district mostly generates income through agriculture. The total population of the Dembecha district was 175,520 in 2022. There were 40,818 households in the district. In 2022, the estimated number of children between 12 and 59 months old in the district was 18,309 ( 24 ). Section title: Study design, period, and population Educational score: 3.2154719829559326 Domain: biomedical Document type: Study Language: en A community-based cross-sectional study design was employed from April 19 to May 25, 2022. The study population was all randomly selected children aged 12–59 months in Dembecha district, during the study period. All children aged 12–59 months in the randomly selected kebeles (the smallest administrative unit in Ethiopia) during data the collection period were included in the study. Children with visual or hearing impairment were excluded from the study since they could not perform the specific developmental domains during the assessment. Section title: Sample size determination and sampling procedures Educational score: 3.315864324569702 Domain: biomedical Document type: Study Language: en The sample size was calculated using a single population proportion formula by considering 29.4% of the developmental delay taken from a previous study in Gurage Zone, Southwest Ethiopia ( 25 ) and 95% confidence interval, 5% marginal error, a design effect of 2, and 10% non-response rate which resulted in 702. Section title: Sample size determination and sampling procedures Educational score: 3.507539749145508 Domain: biomedical Document type: Study Language: en A multistage sampling technique was used to select the study participants. First, one urban kebele and seven rural kebeles were selected using a simple random sampling method. Then, the samples were distributed proportionally based on size. Finally, participants in each kebeles were selected using a systematic sampling technique after calculating the sampling interval for each kebeles The sample frame was the list of households with children 12–59 months of age. A list of households with children was obtained from a family folder, found in the health post. In households with two children less than 5 years of age, one was selected randomly by lottery. A revisit of three times was made in a case where eligible respondents were not available by the time of data collection and those who were not available after the revisit were considered as non-respondent. Section title: Study variables Educational score: 2.0046515464782715 Domain: biomedical Document type: Study Language: en The dependent variable was the child’s developmental status categorized as normal or delayed. Section title: Study variables Educational score: 1.7422934770584106 Domain: biomedical Document type: Study Language: nl The independent variables were: Section title: Operational definitions Educational score: 3.1827809810638428 Domain: biomedical Document type: Study Language: en Developmental delay : It was defined as a child whose ASQ-3 score fell below the cut-off points for their age in any ASQ-3 domains. The five critical domains in a child’s development are communication, gross motor, fine motor, problem-solving, and personal social ( 9 , 26 ) ( Table 1 ). Section title: Operational definitions Educational score: 2.6513795852661133 Domain: biomedical Document type: Other Language: en Stunting : Children with a height-for-age at Z-score below −2 SD of the median value of the World Health Organization (WHO) international standard reference ( 27 ). Section title: Operational definitions Educational score: 2.866255521774292 Domain: biomedical Document type: Other Language: en Wasting : Children with a weight-for-height Z-score below −2 SD of the median value of the WHO international standard reference ( 27 ). Section title: Operational definitions Educational score: 2.6397194862365723 Domain: biomedical Document type: Other Language: en Underweight : Children with weight-for-age Z-score below −2 SD of the median value of the WHO international standard reference ( 27 ). Section title: Operational definitions Educational score: 2.515150547027588 Domain: biomedical Document type: Other Language: en Minimum dietary diversity : The percentage of children 12–59 months who received foods from 4 or more food groups during the previous day. The seven food groups used for tabulation of this indicator are grains, roots, and tubers; legumes and nuts; dairy products (milk, yogurt, and cheese); flesh foods (meat, fish, poultry, and liver/organ meats); eggs; vitamin A-rich fruits and vegetables; and other fruits and vegetables ( 28 ). Section title: Operational definitions Educational score: 2.4318344593048096 Domain: biomedical Document type: Other Language: en Complementary feeding : The process of introducing solid, semi-solid, or soft foods in addition to breast milk at 6 months of age ( 28 ). Section title: Operational definitions Educational score: 2.1000778675079346 Domain: biomedical Document type: Other Language: en Minimum meal frequency : Percentage of children 12–59 months of age who consumed solid, semi-solid, or soft foods at least the minimum number of times during the previous day. The minimum number of times was 4 times of meals per day for 12–59 months of children ( 28 ). Section title: Data collection tool and procedure Educational score: 2.4472429752349854 Domain: biomedical Document type: Study Language: en A structured paper-based questionnaire was used to collect data on socio-demographic, maternal-related factors, child health-related factors, nutritional variables, and food access at the household level. It was constructed by adapting and modifying the Ethiopia Mini Demographic and Health Survey ( 29 ) 2019 and previous research on a similar topic ( 25 , 30 ). First, the English version questionnaire was prepared. Then it was translated to the native language Amharic and was back translated to the English language. Section title: Data collection tool and procedure Educational score: 3.8703577518463135 Domain: biomedical Document type: Study Language: en The child development assessment was done using the ASQ-3. The ASQ-3 has five subscales: communication, gross motor, fine motor, problem-solving, and personal-social. The ASQ-3 was answered with 10 points for “yes,” 5 points for “sometimes,” and 0 points for “not at all.” Each child was asked 30 questions and assessed out of 300 scores; and six questions out of 60 scores for each development milestone. Each domain was classified into three (high risk for developmental delay, needs monitoring, and on schedule) for each age category based on ASQ-3. Finally, child development was categorized as developmental delay and normal development (needs monitoring and on schedule). Developmental delay refers to a high risk of developmental delay whereas normal development refers to a low-to-moderate risk ( 9 , 25 , 26 ) ( Table 1 ). Section title: Data collection tool and procedure Educational score: 2.8275129795074463 Domain: biomedical Document type: Study Language: en The ASQ-3 had a validity of 0.83–0.88, reliability of 0.90–0.94, sensitivity of 0.38–0.91, and specificity of 0.39–0.95 ( 26 , 31 ). This tool was used in a study conducted in the Wolaita Sodo and Gurage zones in South Ethiopia. Also, the ASQ-3 developmental assessment tool was used by 56.3% of health professionals who were working at public hospitals in Addis Ababa, Ethiopia, 2018 ( 32 ). Section title: Data collection tool and procedure Educational score: 2.562201738357544 Domain: biomedical Document type: Study Language: en Children’s dietary diversity was assessed with 7 food groups by using the WHO infant and young child feeding indicator assessment tool with some modifications to fit the context based on the last 24-h recall method ( 28 ). Section title: Data collection tool and procedure Educational score: 1.9358000755310059 Domain: biomedical Document type: Study Language: en Data was collected from caregivers or mothers of the children by two public health professionals and three health extension workers who could communicate well in the local language. The face-to-face interviews were used to collect data for socio-demographic factors and child-related factors. However, most parts of the developmental questions were assessed when a child performed or failed the activity required. The supervisors had oversight over the data collection process and the data collectors and supervisors had regular meetings with the principal investigator at the end of each day of the data collection period. Section title: Data collection tool and procedure Educational score: 4.005366325378418 Domain: biomedical Document type: Study Language: en Anthropometric measurements for height/length were collected as per the World Health Organization (WHO) guidelines. A portable stadiometer was used to measure the height of older children (above 2 years) and a calibrated length board was used for younger children (below 2 years). Older children were measured in a standing position, while younger children below 2 years old were measured in a recumbent position. Children’s height and weight were measured without shoes, hats, and hair ornaments. When measuring height, a child’s head, shoulders, buttocks, and heels were attached to the vertical surface of the stadiometer. The height measurement was recorded to the nearest 0.1 cm. The weight of each child was measured while barefoot and light clothing and recorded to the nearest 0.1 kg using weighing scales ( 33 ). All variables apart from anthropometric measurements and observed ASQ-3 measures were self-reported by the mother/caregiver of a child. Section title: Data quality assurance Educational score: 2.4147331714630127 Domain: biomedical Document type: Study Language: en The quality of the data was maintained by translating the English version questionnaire into the Amharic language, then it was translated back to English to check its consistency. To assess the appropriateness of wording, clarity of the questions, and respondent reaction to the questions, a pre-test was conducted on 5% of the calculated sample size in the Dega Damot district which had similar basic socio-economic characteristics, and the necessary amendments were made. Before data collection, data collectors and supervisors were trained for 1 day by the principal investigator about the overall purpose of the study, how to approach the study participants, and the way to collect data. During the data collection time, close supervision and monitoring were carried out by supervisors and the principal investigator to ensure the quality of the data. Finally, the supervisor and investigator checked the data for accuracy, clarity, consistency, and completeness daily. Section title: Data processing and analysis Educational score: 4.097357273101807 Domain: biomedical Document type: Study Language: en The collected data was checked for completeness. The pre-coded data was entered into Epi data version 3.1 statistical software. Then the data was exported to SPSS version 25 for analysis. Descriptive analysis such as frequency, percentage, mean and standard deviation was computed, and the results were presented using texts, crosstabs, tables, and figures. The child’s developmental status was computed for each milestone as delayed and normal development and the five milestones were combined to determine the child’s overall developmental status. Finally, child development was categorized as developmental delay and normal development. WHO Anthro version 1.0.4 software was used to convert the anthropometric measure weight, height, and age value into Z-score of the indices HAZ, WAZ, and WHZ. Children whose height-for-age, weight-for-height, and weight-for-age < −2 SD from the median of the reference population were considered stunted, wasted, and underweight, respectively. Binary logistic regression was performed to identify factors significantly associated with child development delay. Those variables with a p -value <0.25 in bivariable analysis were entered into the multivariable analysis to control for possible confounding variables and to examine the association. The strength of association was measured using the adjusted odds ratio with 95% confidence intervals. Statistical significance was declared at a p -value of <0.05. A good fitness of the model was observed from the Hosmer Lemeshow test. The absence of multicollinearity was checked by using variance inflation factors and tolerance. The variables variance inflation factor and tolerance were < 10 and > 0.1, respectively. Section title: Ethical consideration Educational score: 1.768447756767273 Domain: biomedical Document type: Study Language: en An ethical clearance letter was obtained from the Research Ethics Committee of the College of Health Science of Debre Markos University (Reference Number: HSC/R/C/Ser/PG/Co/118/11/14). A written official letter along with the ethical clearance was submitted to Dembecha District Health Office by the principal investigator to get permission from the Health office before the data collection. In addition, written informed consent was taken from the mothers of the children. While conducting the study, children who were found to be malnourished, their mothers were informed and advised about the measures to be taken, and were linked to the nearby public health services for treatment and support. The obtained information was kept confidential by locking it with a password. Section title: Results Educational score: 2.62388277053833 Domain: biomedical Document type: Study Language: en This study approached 702 mothers with children aged 12–59 months in Dembecha district, with a 98.00% response rate (688 mothers with children). The average age of the respondents was 31.28 years, and 274 (39.80%) were between the ages of 25 and 34. The majority of respondents were from rural areas (73.7%), Ethiopian Orthodox Tewahedo church followers (93.6%), married (85.6%), and had a family size of less than five (52.6%). About 85.2% of mothers were housewives, while 74.3 and 34.9% of fathers were farmers and had a primary education, respectively ( Table 2 ). Section title: Maternal obstetric and healthcare-related characteristics Educational score: 2.428990602493286 Domain: biomedical Document type: Study Language: en About 96.51% of children were born in a health facility. The majority of children (65.0%) were born at term. More than two-thirds (69.9%) of the children were born via spontaneous vaginal delivery. Most of the mother (95.4%) did not develop hypertension during pregnancy. Half of the mothers (51.3%) had no history of illness or disease during pregnancy. More than half of the mothers (57.9%) drank alcohol while pregnant ( Table 3 ). Section title: Socio-demographic, feeding practice and nutritional status of children Educational score: 2.774482488632202 Domain: biomedical Document type: Study Language: en Three hundred eighty-four (55.8%) children were females. The mean age of the children was 31.0 months (SD = 12.4 months). Two-thirds (66.6%) of the children were toddlers aged 1–3 years. More than three-fourths (87.8%) of children were born with normal birth weight. In terms of feeding, 373 (54.2%) began complementary feeding at the age of 6 months. Less than half of children (41.0%) eat less than four times per day. In the previous 24 h, 57.0% of children met the minimum dietary diversity (four food groups; Table 4 ). Section title: Socio-demographic, feeding practice and nutritional status of children Educational score: 2.9337551593780518 Domain: biomedical Document type: Study Language: en Concerning the nutritional status of children, about 27.8% (95% CI: 24.4, 31.3%), 9.2% (95% CI: 7.1, 11.6%), and 4.5% (95% CI: 3.1, 6.3%) were stunted, underweight, and wasted, respectively . Section title: Prevalence of childhood developmental delay Educational score: 4.039217948913574 Domain: biomedical Document type: Study Language: en The mean ASQ-3 score was 155.4, with a standard deviation of 25.7, a minimum score of 80, and a maximum of 270. The mean ASQ-3 score for each domain ranged from 27.2–35.9, with a standard deviation from 5.5–8.2, and a range from 10 to 55 for communication,10 to 60 for fine motor, and 15 to 60 for the remaining of the milestones. This study found that 26.7% (95% CI: 23.5, 30.2%) of children have developmental delays. It is expressed as communication delay (17.2%), gross motor delay (20.6%), fine motor delay (17.4%), problem-solving delay (16.7%), and personal social delay (18.2%). The remaining 73.3% with 95%CI (69.8, 76.5%) were on normal development to their age . Section title: Prevalence of childhood developmental delay Educational score: 2.455826997756958 Domain: biomedical Document type: Study Language: en About 28 (4.1%; 95% CI: 2.7, 5.8%) children had developmental delays in all the five developmental domains. Section title: Factors associated with childhood developmental delay Educational score: 3.911308526992798 Domain: biomedical Document type: Study Language: en Maternal age, marital status, weight at birth, gestational age, time of initiation of complementary feeding, birth order, place of delivery, mode of delivery, child age, stunting, underweight, parity, meal frequency, dietary diversity, and family size were associated with child development delay in bivariable logistic regression analysis and identified as candidates for multivariable logistic regression analysis ( Table 6 ). Section title: Factors associated with childhood developmental delay Educational score: 4.022467136383057 Domain: biomedical Document type: Study Language: en In multivariable logistic regression analysis; toddler age of the child, LBW, cesarean section mode of delivery, preterm delivery, initiation of complementary feeding before 6 months, stunting, inadequate meal frequency, and inadequate dietary diversity were significantly associated with developmental delay at p -value of 0.05 level of significance ( Table 6 ). Section title: Factors associated with childhood developmental delay Educational score: 4.0624470710754395 Domain: biomedical Document type: Study Language: en Children born with a LBW were nearly 5 times more likely to experience developmental delay than children with a normal birth weight (AOR = 4.90; 95% CI: 2.14, 11.48). Toddlers (1–3 years) were 2.6 more likely to experience developmental delay than children who were in the age of preschool (3–5) years (AOR = 2.60; 95% CI: 1.42, 4.87). Children born by cesarean section were nearly 9 times more likely to have developmental delay (AOR = 8.60; 95% CI: 3.93, 18.65; Table 6 ). Section title: Factors associated with childhood developmental delay Educational score: 4.0630412101745605 Domain: biomedical Document type: Study Language: en Children who started complementary feeding before 6 months of age were 8.4 times more likely to have developmental delay than children who started at 6 months (AOR = 8.40; 95% CI: 3.61, 19.63). Preterm children were 2.5 times more likely to experience developmental delay compared to children who were delivered at term (AOR = 2.50; 95% CI: 1.28, 4.74). Children who had meal frequency of <4 times were 3 times more likely to develop developmental delay compared to children who had meal frequency of ≥4 times (AOR = 3.20; 95% CI: 1.74, 5.94; Table 6 ). Section title: Factors associated with childhood developmental delay Educational score: 3.9837706089019775 Domain: biomedical Document type: Study Language: en Children who had inadequate dietary diversity were 3 times more likely to develop developmental delay compared to children who received more than four food groups (AOR = 3.10; 95% CI: 1.68, 5.85). The odds of developmental delay among stunted children were nearly 3 times higher as compared to normal children (AOR = 2.90; 95% CI: 1.67, 5.22; Table 6 ). Section title: Discussion Educational score: 3.7107045650482178 Domain: biomedical Document type: Study Language: en This study aimed to assess child developmental delay and its associated factors. This study found that the prevalence of child developmental delay was 26.7%. Developmental delay among children was associated with toddler child age, LBW, cesarean section mode of delivery, preterm delivery, initiation of complementary feeding before 6 months, stunting, inadequate meal frequency, and inadequate dietary diversity. Section title: Discussion Educational score: 2.202918767929077 Domain: biomedical Document type: Study Language: en The prevalence of child developmental delay in this study was in line with studies conducted in Ethiopia (29.4%) ( 25 ) and Iran (26.3%) ( 34 ). Section title: Discussion Educational score: 4.051944255828857 Domain: biomedical Document type: Study Language: en However, the prevalence of child developmental delay in this study was higher compared to other studies conducted in Ethiopia (19%) ( 35 ), Central Iran (11.8%) ( 12 ), and the global prevalence (8.4%) ( 11 ). The discrepancy might be due to the better socio-economic status, and the child health care system in Iran. The difference from the previous Ethiopian study might be attributed to the high prevalence of malnutrition in the current study area ( 30 ), which results in developmental delay ( 35 ). On the contrary, the prevalence of child developmental delay in this study was lower compared to studies conducted in Nepal (56.4%) ( 5 ), South Africa (55.2%) ( 36 ), and Nigeria (35.4%) ( 37 ). The possible reasons for this variation might be attributed to several factors including the study method used, the different socio-economic classes of participants, differences in study settings, and different tools for assessing developmental milestones. In addition, the high prevalence of child developmental delay in Nepal might be due to the inclusion of urban slum areas in their study. Children living in slum areas have a higher risk of undernutrition ( 38 , 39 ), and Human Immuno-deficiency virus (HIV) infection ( 40 ), ultimately contributing to child developmental delay. Section title: Discussion Educational score: 4.01833438873291 Domain: biomedical Document type: Study Language: en Moreover, the prevalence of developmental delay was lower compared to the national estimate of Ethiopia (59%) ( 21 ) and the Lancet 2016 report from the world which was 43% of children under 5 years of age living in low- and middle-income countries being at risk of suboptimal development ( 4 ). The difference from the Lancet Report is attributed to the inclusion of a wide population of the globe. The current study shows children aged 1–3 years had higher odds of developmental delay as compared to 3-5-years-old children. This finding is supported by a study in Pakistan ( 41 ). The probable reason might be infants and toddlers grow and develop rapidly in the first 2 years, making them particularly vulnerable or influenced by nutritional inadequacies, and environmental and socio-demographic variables. A vital window of opportunity for ensuring children’s proper growth and development through adequate nutrition occurs throughout the first 2 years of life. It can also be due to the cultural aspect that young children are mostly kept indoors and therefore have less exposure and stimulation. Section title: Discussion Educational score: 4.0536699295043945 Domain: biomedical Document type: Study Language: en In this study, children with LBW have a higher likelihood of developing developmental delay compared to children with normal birth weight which is similar to a study done in West Bengal ( 42 ). Because they are more likely to have neuro-developmental abnormalities, children with lower birth weights are likely to have a higher chance of experiencing developmental delays. It is reported that approximately 10% of the extremely LBW (< 1,000 grams) children will develop cerebral palsy which is a disorder of movement, posture, and balance ( 43 ). Also, a 32% rate of cerebral palsy is found in those infants weighing less than 1,500 grams ( 44 ). Low birth weight remains to be a risk factor for developmental delay which can easily be prevented by regular monitoring and better nutrition. Section title: Discussion Educational score: 4.088194847106934 Domain: biomedical Document type: Study Language: en In this study, the cesarean section mode of delivery had higher odds of developmental delay in children which was similar to other studies done in Brazil ( 45 ). The possible reason for this increment might be associated with the absence of contact with the mother’s natural bacterial flora during a cesarean section delivery. Vaginally delivered babies are exposed to microbes residing in the maternal birth canal. Microbial colonization of the infant gastrointestinal tract is important for the development of gut immunology, brain-gut-axis, and nervous system, influencing brain function, and behavior ( 46 , 47 ). In addition, neonates delivered through cesarean section have a high risk of birth asphyxia ( 48 , 49 ), which contributes to child developmental delay ( 50 ). Section title: Discussion Educational score: 4.072727203369141 Domain: biomedical Document type: Study Language: en Premature birth in the current study was associated with a higher risk of developmental delay compared to term birth, which is similar to other studies conducted in Turkey ( 51 ), North India ( 52 ), and Southwest Ethiopia ( 25 ). The probable explanation for the increase of child developmental delay in preterm children is due to an increased risk of hypoxic–ischemic events that often lead to severe cognitive developmental delays, or motor impairments ( 53 ). Preterm children are also at risk of intraventricular bleeds and infections likely to result in neural injury as well ( 54 ). Section title: Discussion Educational score: 4.0832977294921875 Domain: biomedical Document type: Study Language: en This study also showed that children who started complementary feeding before 6 months had increased odds of developmental delay. The evidence was similar to the findings from Saudi Arabia ( 35 , 55 ), and Ethiopia ( 25 ). Babies who start complementary feeding early have higher rates of iron deficiency anemia because it can decrease iron absorption from breast milk. Iron is an important element for brain growth and development as demonstrated in the fetuses and neonates ( 56 ). In addition, feeding other than breast milk causes early satiety which interferes with the infant’s feeding behavior, decreases the frequency of breastfeeding, or reduces breast milk production. Since human milk contains fatty acids like arachidonic acid and docosahexaenoic acid, it is important for the appropriate development of a baby’s organs, tissues, and brain development. The essential fatty acids enhance the production and activity of various immune cells, including lymphocytes and phagocytes ( 57 , 58 ). Thus, exclusive breastfeeding for up to 6 months decreases the risk of infections in infants compared to early initiation of complementary feeding for infants ( 59 ). Section title: Discussion Educational score: 4.130740642547607 Domain: biomedical Document type: Study Language: en The current study showed that stunted children had higher odds of developmental delay. This finding is consistent with the study conducted in Ethiopia ( 25 , 35 ), Nigeria ( 37 ), and Nepal ( 5 ). This is because malnutrition results in tissue damage, growth retardation, disorderly differentiation, reduction in synapses and synaptic neurotransmitters, delayed myelination, and reduced overall development of the brain. Inadequate brain growth explains why malnourished children suffer from behavioral and cognitive deficits, including slower language and fine motor development. Because they are lethargic and apathetic, malnourished children have a problem understanding the information and they are less interested in their surroundings than well-nourished children. This results in delayed social interaction skills ( 60 ). Children who are malnourished also have deficiencies in micronutrients, like calcium and vitamin D, which are critical for the health of their skeletal muscles. Therefore, a lack of certain micronutrients may have an impact on motor abilities. Nutritional insufficiencies at an acute stage may damage the cognitive profile and entire auditory system in children, resulting in verbal and written language problems ( 37 ). Section title: Discussion Educational score: 3.7481255531311035 Domain: biomedical Document type: Study Language: en Children with inadequate meal frequency (<4 times a day) have increased odds of child developmental delay. This evidence was supported by findings from Ethiopia ( 35 ) and China ( 61 ). In order to promote brain growth, which is marked by intense myelination throughout the first 5 years of life, children need a lot of energy from food. Failure to meet energy needs may lead to changes in metabolism in the brain and decreases in muscle strength, thus affecting the child’s ability to develop normal gross motor skills ( 61 ). Section title: Discussion Educational score: 4.095248699188232 Domain: biomedical Document type: Study Language: en Furthermore, the current study also shows that low dietary diversification had higher odds of child developmental delay. This result is comparable with the study findings in ( 35 ) Ethiopia ( 25 ), and China ( 61 ). The relationship between dietary diversity and child developmental outcomes might be explained by several mechanisms, including increased intake of micronutrients and protein, greater amounts and variety of psychosocial stimulation, and building muscles for enhanced motor skills, physical activity, and social function, which may, in turn, enrich the child’s interaction with and exploration of the environment, ultimately contributing to the development of problem-solving skills. The increased risk for developmental delay due to low diversification is that low dietary diversity causes nutrient deficiency like protein, energy, fats, iron, zinc, copper, iodine, selenium, vitamin A, choline, and folate ( 62 ). Therefore, brain development, both structural and functional, is highly dependent on an adequate supply of protein and micronutrients. Section title: Discussion Educational score: 3.4787888526916504 Domain: biomedical Document type: Study Language: en The findings should be interpreted considering the following limitations; Recall bias might be introduced during the collection of child birth weight, age at initiation of complementary feeding, and gestational age since it depends on the respondents’ memory. In addition, we did not collect data on the breastfeeding versus formula-feeding status of a child, maternal height, and maternal mental health status. As a result, the study findings have not been adjusted for these variables. Thus, future researchers shall consider these factors in their study. Furthermore, because the study was cross-sectional, it was unable to demonstrate a cause-and-effect relationship. Moreover, there have not been studies exploring the reliability and validity of the ASQ-3 for Ethiopia, as a result, the results should be interpreted with this limitation in mind. Section title: Conclusion Educational score: 3.9456489086151123 Domain: biomedical Document type: Study Language: en In conclusion, the prevalence of child developmental delay was high in Dembecha district as compared to the global prevalence. Child development delay was associated with toddler child age, LBW, cesarean section mode of delivery, preterm delivery, initiating complementary feeding before 6 months, stunting, inadequate meal frequency, and inadequate dietary diversity. Therefore, preventing preterm delivery, initiation of complementary feeding before 6 months, stunting and achieving the minimum meal frequency, and minimum dietary diversity are recommended to prevent developmental delay among children aged 12–59 months. | Review | biomedical | en | 0.999999 |
PMC11695296 | Section title: 1 Introduction Educational score: 3.9114081859588623 Domain: biomedical Document type: Study Language: en Nickel is a silver-white, ferromagnetic, corrosion-resistant metal element that is widely distributed in the lungs, liver, kidneys, and other tissues and organs. It is also one of the essential trace elements in the human body. A deficiency of nickel can lead to a decrease in enzyme activity and can impact human growth and development ( 1 ). However, excessive exposure to nickel can pose health hazards such as neurasthenic reactions, inflammatory responses, and oxidative stress injuries ( 2 , 3 ). Studies have indicated that occupational populations exposed to high levels of nickel for prolonged periods are at an increased risk of developing lung cancer, nasopharyngeal cancer, and occupational asthma ( 4 – 10 ). Section title: 1 Introduction Educational score: 4.165510654449463 Domain: biomedical Document type: Study Language: en In 1990, the International Agency for Research on Cancer (IARC) classified nickel compounds as Group I carcinogens and metal nickel as a Group II B carcinogen. Na et al. ( 11 ) conducted a retrospective cohort study on four nickel-receiving factories and mines, revealing an increased risk of lung cancer death among workers in nickel refining and smelting. Pesch et al. ( 12 ) carried out a case-control study within a German occupational population involved in nickel welding, finding a dose-response relationship between nickel exposure and the risk of lung cancer. The mechanism by which nickel exposure induces lung cancer has also been verified. Several studies have demonstrated that molecular mechanisms such as apoptosis and autophagy are induced by nickel exposure ( 13 – 16 ). Other studies have found that epigenetic changes, activation of hypoxia signaling pathways, and production of reactive oxygen species are also implicated in how nickel and its compounds induce cancer at a molecular level ( 17 ). Section title: 1 Introduction Educational score: 3.8175199031829834 Domain: biomedical Document type: Other Language: en Disability-adjusted life years (DALY) serves as an indicator of the disease burden, calculating all healthy years of life lost from the onset to death. This includes years of life lost due to premature death (YLL) and years of healthy life lost due to disability from illness (YLD). The global burden of disease (GBD) database has been in operation since 1988, its purpose is to analyze the global burden of disease, encompassing health burdens related to disease, risk factors, disabilities, and mortality ( 18 ). Section title: 1 Introduction Educational score: 3.995012044906616 Domain: biomedical Document type: Study Language: en China is a leading global producer of nickel compounds, according to the 2022 report on the prevalence of malignant tumors in China, lung cancer has the highest incidence among all malignant tumors ( 19 ). However, there is a lack of analysis on the burden of lung cancer related to nickel exposure. Therefore, this paper aims to extract mortality and disease burden data related to nickel-induced lung cancer from the GBD database. The study will analyze the disease burden of nickel-related lung cancer from 1990 to 2019 and its changing trend. Additionally, relevant software will be used to predict the disease burden of nickel-related lung cancer over the next 15 years. The findings aim to provide suggestions for developing occupational disease prevention measures and improving the quality of life for current patients. Section title: 2.1 Study data Educational score: 3.3881735801696777 Domain: biomedical Document type: Study Language: en Data from this study is obtained from the GBD2019 database and the global health data exchange (GHDx) website, 1 which provides access to public data. The Institute for Health Metrics and Evaluation (IHME) systematically estimates the incidence, prevalence, mortality, DALY, YLL, and YLD for 369 diseases and injuries in 204 countries and territories ( 20 , 21 ). The cancer codes used in the GBD correspond to the International Statistical Classification of Diseases and Related Health Problems (Tenth Revision) (ICD-10) ( 22 ). Section title: 2.2 Statistical analysis Educational score: 4.009974479675293 Domain: biomedical Document type: Study Language: en In this study, the number of deaths and standardized mortality were chosen to reflect the risk of death in a specific population during a certain period. YLL represents the years of life lost, while YLD reflects years lived with disability. DALY was utilized as an indicator of disease burden for nickel-associated lung cancer, comprehensively reflecting the impact of life loss caused by both YLL and YLD. Section title: 2.2 Statistical analysis Educational score: 3.5435993671417236 Domain: biomedical Document type: Study Language: en ASR stands for the age-standardized rate, while ASMR refers to the age-standardized mortality rate and ASDR represents the age-standardized DALY rate. The 95% uncertainty interval (UIs) was employed to estimate the number of deaths and other indicators, while 100% annual change (APC) and average annual change percentage (AAPC) were used to analyze trends in statistical indicators such as ASDR (abbreviations are utilized following the text). Joinpoint software was utilized for calculating and statistically analyzing AAPC, with R software being used for data cleaning, visual display, and testing at a significance level α = 0.05. Section title: 3.1 Mortality and DALY in nickel-associated lung cancer in China and globally Educational score: 4.1474995613098145 Domain: biomedical Document type: Study Language: en We conducted an analysis of the trends in nickel-associated lung cancer deaths and DALY in China and globally from 1990 to 2019. During this period, the total number of nickel-associated lung cancer deaths in China was 83,720, accounting for 40.2% of the global nickel-associated lung cancer deaths. The death rate of nickel-associated lung cancer in China increased by 145.8%, rising from 1,668 to 4,100. Globally, nickel-associated lung cancer deaths increased from 5,248 to 9,330, representing a rise of 77.8% . Furthermore, the DALY caused by nickel-associated lung cancer in China increased from 52,043 person-years to 114,606 person-years, marking an increase of 120.2%. The global DALY due to nickel-associated lung cancer increased from 158,519 person-years to 260,986 person-years, representing a significant increase of 64.6% . As depicted in Figure 2 , the ASMR of nickel-associated lung cancer in the Chinese population showed an initial increase followed by a decrease during the period of 1990–2019 . Conversely, there was an overall downward trend globally . The ASDR of nickel-associated lung cancer in China exhibited an initial increase followed by a decrease with no statistical significance , while there was an overall downward trend globally . Section title: 3.2 Analysis of the disease burden caused by lung cancer attributable to nickel exposure Educational score: 4.149693489074707 Domain: biomedical Document type: Study Language: en From an alternative perspective, the YLL and YLD caused by nickel-associated lung cancer in the Chinese population increased from 51,625 person-years and 418 person-years to 113,365 person-years and 1,241 person-years, respectively, between 1990 and 2019 . The global population saw an increase from 157,118 and 1,402 to 258,195 and 2,790 respectively. It is worth noting that the growth rate of China was higher than that of the world. The age-standardized YLD rate (ASYLDR) increased from 0.044/100,000 to 0.057/100,000 with AAPC = 0.9% ( P < 0.001), while the global AAPC decreased from 0.033/100,000 to 0.033/100,000 with AAPC = −0.0% ( P = 0.737) . The age-standardized YLL rate (ASYLLR) decreased from 5.300/100,000 in 1990 to 5.196/100,000 in 2019, with an AAPC of −0.1% ( P = 0.408). The global AAPC also showed a decrease from 3.673/100,000 to 3.037/100,000, with an AAPC of −0.7% ( P < 0.001) . The increase in YLD/YLL for nickel-associated lung cancer in the Chinese population (35.1%) was higher than that in the global population (21.2%), and by 2018 the YLD/YLL in the Chinese population had surpassed that of the global population for the first time . Section title: 3.3 Trends in the global burden of disease by region Educational score: 3.974903106689453 Domain: biomedical Document type: Study Language: en From a global perspective, there is significant variation in the changes in the burden of nickel-associated lung cancer disease from 1990 to 2019 across different countries. The proportion of lung cancer deaths attributable to nickel has seen substantial increases in Egypt, Pakistan, and Ethiopia, ranging from 0.10 to 0.76%. Conversely, countries such as Russia, Sweden, and Saudi Arabia have experienced notable declines in the nickel attributable proportion of lung cancer deaths, ranging from 0.19 to 0.66% . When considering the nickel attribution ratio in DALY caused by lung cancer, it is evident that Egypt, Pakistan, Vietnam and other countries have also witnessed significant increases ranging from 0.09 to 0.80%. On the other hand, Russia, Sweden and Saudi Arabia stand out for experiencing the highest declines in this ratio ranging from 0.17 to 0.65% . Section title: 3.3 Trends in the global burden of disease by region Educational score: 3.7508459091186523 Domain: biomedical Document type: Study Language: en From the perspective of ASMR for nickel-associated lung cancer, countries such as India, Pakistan, and Egypt have shown the largest annual average increase, ranging from 0.26 to 1.10%. Conversely, the United States, Russia, and South Africa experienced the largest annual average declines in this regard, with percentages ranging from 0.32 to 0.70% . In terms of ASDR for nickel-associated lung cancer, Indonesia, Egypt, Serbia and other countries demonstrated the largest annual average increase at a range of 0.12–0.98%. On the other hand, countries such as the United States, Canada and Russia experienced significant annual average declines in ASDR of nickel-associated lung cancer at a range of 0.34 to 0.72% . Overall trends indicate an upward trend in African and Southeast Asian countries while European and East Asian countries show a downward trend for nickel-associated lung cancer rates. Section title: 3.4 Age and gender distribution of the burden of lung cancer attributable to nickel exposure Educational score: 4.124988555908203 Domain: biomedical Document type: Study Language: en In the analysis of the burden of nickel-associated lung cancer by sex, it was observed that the ASMR and ASDR of nickel-associated lung cancer in China and the global population exhibited a pattern of initially increasing and then decreasing with advancing age, with higher rates in men than in women. The ASMR of nickel-associated lung cancer in Chinese men and women peaked in the 60–64 age group in 1990 and in the 70–74 age group in 2019 . In the global population, the ASMR of nickel-associated lung cancer reached its zenith in 1990 among women aged 65–69 and men aged 60–64. However, by 2019, both men and women showed a peak ASMR in the 70–74 age group . Similarly, the ASDR of nickel-associated lung cancer among Chinese men and women also peaked between the ages of sixty to sixty-four during both time periods ; while globally, the ASDR reached its pinnacle at ages between sixty to sixty-four years old during both time periods . Section title: 3.5 Prediction of the burden of lung cancer attributable to nickel exposure in the next 15 years Educational score: 4.110718727111816 Domain: biomedical Document type: Study Language: en This study is based on the Nordpred model to predict the ASMR and ASDR of nickel-associated lung cancer in China and globally from 2020 to 2035. The results indicate that deaths related to nickel-associated lung cancer in China and globally are expected to reach their peak in 2027, with 4,087 and 9,874 cases, respectively . The DALY of nickel-associated lung cancer in both China and worldwide shows a pattern of initial increase followed by decrease, reaching its peak in 2027 at 114,942 person-years and 268,731 person-years, respectively . Table 5 and Figure 9A demonstrate that the ASMR of nickel-associated lung cancer in China exhibits an initial increase followed by decrease. Globally, the ASMR for nickel-associated lung cancer shows an initial increase followed by stabilization. Table 5 and Figure 9B show that the ASDR of nickel-associated lung cancer in China demonstrates an increasing trend; however, globally it shows a decreasing trend. Section title: 4 Discussion Educational score: 4.064511299133301 Domain: biomedical Document type: Study Language: en Based on the death toll, ASMR, DALY and other disease burden indicators in China and the world in GBD2019 database, this study analyzed and predicted the disease burden data and its changing trend of nickel-associated lung cancer. The normalized DALY, YLL and YLD rates of nickel-associated lung cancer in the Chinese population were calculated using the average age structure of the world population as the standard population, so as to form a comparable result with the global population. Compared with previous studies that mainly focused on the disease burden of lung cancer caused by risk factors such as tobacco and air pollution, this study focused on the disease burden of occupational nickel-exposed populations, paying more attention to occupational populations. It supplemented current neglected studies on the disease burden of nickel-associated lung cancer. Section title: 4 Discussion Educational score: 4.074620246887207 Domain: biomedical Document type: Study Language: en In 2019, the DALY of nickel-associated lung cancer in China accounted for 0.03% of the total disease burden, represented 43.91% of the global nickel-associated lung cancer disease burden ( 23 ). The disease burden of lung cancer caused by occupational nickel exposure is relatively small compared to other widely distributed risk factors for lung cancer ( 24 , 25 ). However, it is important to note that our study found an increasing number of nickel-associated lung cancer deaths and ASDR in China and globally, with China experiencing a greater increase, the potential risk of developing lung cancer due to exposure to nickel cannot be overlooked, particularly within the context of China. On the contrary, there were no significant changes in the ASMR and ASDR of nickel-associated lung cancer, indicating there was no significant change in the impact of nickel on lung cancer within the population. The increasing burden of nickel-associated lung cancer is attributed to the aging age structure of patients. Section title: 4 Discussion Educational score: 4.073540687561035 Domain: biomedical Document type: Study Language: en The findings indicate that from 1990 to 2019, there was a consistent decrease in ASYLLR and an increase in YLD/YLL for both the Chinese and global populations affected by nickel-associated lung cancer. This suggests a reduction in the premature mortality impact of nickel-associated lung cancer, but an increase in the burden of disability. Furthermore, analysis of ASMR and ASDR for nickel-associated lung cancer in the Chinese population revealed a shift toward older age groups between 1990 and 2019. The reasons for this trend are twofold. Firstly, there is the acceleration of China’s population aging process ( 26 , 27 ). Secondly, there has been an improvement in occupational health protection and medical care levels, as well as the implementation of effective three-level prevention measures ( 28 – 30 ). These factors have led to a reduction in disease mortality rates and an extension of patient survival times. However, a new challenge we face is how to improve the quality of life for patients. Section title: 4 Discussion Educational score: 4.136108875274658 Domain: biomedical Document type: Study Language: en Then, we projected the future trend of ASMR and ASDR in nickel-associated lung cancer in the Chinese population. It is anticipated that ASMR will initially increase and then decrease over the next 15 years, while ASDR is expected to show a continuous upward trend. At present, China’s new energy industry is developing rapidly, the scale of nickel industry is expanding, the occupational exposure of nickel and the general population exposure are increasing ( 31 , 32 ), and China is in the aging process, the ASDR of nickel-associated lung cancer may highlight the rising trend. Compared with a general lung cancer risk analysis, this study focuses more on the harm caused by nickel as an occupational exposure factor to the population, which is also the key population exposed to nickel. With the robust development of the new energy industry in recent years, nickel has found extensive applications across various industrial sectors, including electroplating, battery manufacturing, and aerospace. This widespread utilization can be attributed to its exceptional corrosion resistance, physical strength, and distinctive ferromagnetic properties. Therefore, we contend that to effectively manage the disease burden of nickel-associated lung cancer, it is essential to prioritize the protection of occupational nickel-exposed populations. Additionally, it is crucial to address the impact of the aging age structure of patients on nickel-induced lung cancer. Finally, efforts should be made to mitigate the disease burden resulting from disability. Section title: 4 Discussion Educational score: 4.154895782470703 Domain: biomedical Document type: Other Language: en First and foremost, it is essential to implement effective primary prevention strategies aimed at reducing the incidence and mortality associated with nickel-associated lung cancer. Enhance the working environment and monitor nickel concentration levels in the workplace to ensure compliance with national safety standards. Implement advanced ventilation systems and utilize personal protective equipment in the workplace to effectively minimize workers’ direct exposure to nickel. In addition, it is essential to provide regular occupational health training for workers to enhance their awareness of the risks associated with nickel exposure, and user-friendly health education models specifically tailored for older workers should be developed. Second, enhance the routine occupational health assessments for workers exposed to nickel, and ensure effective implementation of secondary prevention measures. Enhance the monitoring and assessment of occupational health, and establish as well as improve personal health records for workers exposed to nickel. In particular, it is essential to increase the frequency of occupational health examinations for older workers. For individuals exhibiting mild symptoms or identified potential risks, early intervention measures such as job reassignment or a reduction in work intensity should be enacted. Third, it is essential to not only focus on the disease itself but also to consider the quality of life of patients with nickel-associated lung cancer, do a good job of tertiary prevention, and reduce the burden of disease. Timely identification of patients’ disabilities and mental health conditions, along with the provision of professional rehabilitation guidance and services for workers affected by disability or illness, is essential to assist them in regaining their physical function to the greatest extent possible. Enhance mental health support and offer psychological counseling to affected workers and their family members in order to alleviate the mental stress induced by the disease. Section title: 4 Discussion Educational score: 2.976267099380493 Domain: biomedical Document type: Study Language: en There are certain limitations to this study. First, all the data used in our study came from GBD 2019, which originated from various sources ( 33 ). Consequently, there may be inconsistencies and incompatibilities between these data, potentially leading to bias. Second, as a measure of disease burden, DALY is restricted to the patient group level and does not evaluate the burden caused by out-patient groups such as family, society, and the state. Finally, these data rely on the calculation of monitoring data, and there may be lags in estimating the current BOD. Section title: 5 Conclusion Educational score: 3.940355062484741 Domain: biomedical Document type: Study Language: en In summary, although the DALY of nickel-associated lung cancer in the Chinese population is relatively low compared with other causes, it accounts for a high proportion globally. Additionally, the age structure of nickel-associated lung cancer patients shows an aging trend, and the ASDR in the Chinese population indicates a potential upward trend when projecting the disease burden of nickel-associated lung cancer over the next 15 years. Therefore, in the future, China should focus on occupational health prevention, strengthen protection for those in occupational contact with nickel, increase basic scientific research on nickel-associated lung cancer, pay attention to improving patients’ quality of life and mental health protection, and reduce the disease burden of nickel-related lung cancer within our population. | Study | biomedical | en | 0.999996 |
PMC11695298 | Section title: Introduction Educational score: 4.446617603302002 Domain: biomedical Document type: Review Language: en Ca 2+ acts as a second messenger in various cellular processes ( 1 , 2 ). Increasing evidence suggests that Ca 2+ signaling is involved in various diseases, including cancer, autoimmune diseases, and viral infections. Mutations, abnormal expression, regulation, or subcellular targeting of Ca 2+ handling/transport proteins in cancer can distort Ca 2+ signaling, dysregulating Ca 2+ -dependent effectors and promoting cancer pathophysiology. Cell proliferation, angiogenesis, invasion, and metastasis are important for tumor development ( 3 ). Abnormal Ca 2+ signaling contributes to malignant phenotype development. To achieve rapid proliferation, increased cell motility and invasion, evasion of cell death, evasion of immune attack, or the formation of new blood vessels, tumors remodel their Ca 2+ signaling networks. Tumorigenic pathways are increasingly linked to changes in the expression or activation of Ca 2+ channels, transport proteins, or Ca 2+ -ATPases ( 4 , 5 ). Section title: Introduction Educational score: 4.439676284790039 Domain: biomedical Document type: Review Language: en Head and neck cancer continues to be a leading cause of cancer-related mortality among newly diagnosed cases globally each year. Despite advancements in treatment, around 40% of newly diagnosed patients succumb to this disease ( 6 ). Head and neck squamous cell carcinoma (HNSCC), the most common malignant tumors in the head and neck region, originate from the mucosal epithelium of the oral cavity, nasopharynx, oropharynx, hypopharynx, and larynx. Oropharyngeal and laryngeal cancers are typically linked to smoking and heavy alcohol use, whereas pharyngeal cancer is increasingly associated with human papillomavirus (HPV) infection ( 7 ). HNSCC is a type of adult cancer. The median age at diagnosis is 66 years for HPV-negative HNSCC, 53 years for HPV-positive HNSCC, and 50 years for EBV-positive HNSCC ( 8 ). Regardless of environmental or viral causes, men have a significantly higher risk of developing various forms of HNSCC than women. The typical symptoms of HNSCC vary based on the primary tumor’s anatomical location and its cause, such as environmental carcinogens, HPV, or EBV ( 9 ). Advances in biotechnology, surgery, and radiation therapy are improving the prognosis of HNSCC patients, particularly with the inclusion of immune checkpoint inhibitors. Ongoing clinical trials and advancements in precision medicine are progressing rapidly ( 7 ). Section title: Introduction Educational score: 4.075538635253906 Domain: biomedical Document type: Review Language: en In the development of treatments for HNSCC we have gained a better understanding of changes in the genome, proteome, microbiome, and metabolome, which helps us move closer to personalized therapy. Our current understanding of the disease not only considers endogenous changes as key factors in tumorigenesis but also takes into account the microenvironmental factors that contribute to carcinogenic mechanisms ( 10 ). There is still a limited understanding of the mechanisms of HNSCC development. Calcium channels were first reported in prostate cancer, where it was found that blocking of calcium channel could affect the progression of cancer ( 11 ). Therefore, reshaping these dysregulated Ca 2+ characteristics could be a potential target for cancer treatment ( 12 ). Recent advances in calcium channel research have provided new directions for more accurately diagnosing and treating HNSCC. This review delineates the mechanisms of calcium channels in HNSCC development, highlighting recent findings on drugs and methods targeting specific calcium channels for HNSCC treatment. Section title: Altered cellular calcium transport systems in HNSCC Educational score: 4.520333290100098 Domain: biomedical Document type: Study Language: en The precise regulation of the calcium transport system governs Ca 2+ movement between intracellular and extracellular spaces, as well as between the cytoplasm and organelles . The plasma membrane calcium transport system comprises calcium release-activated calcium (CRAC) channels, transient receptor potential (TRP) channels and voltage-gated calcium channels (VGCC/Cavs) ( 13 ). These channels facilitate the entry of extracellular Ca 2+ into the cell, leading to elevated intracellular Ca 2+ levels. CRAC channels, representative of store-operated calcium entry (SOCE) channels, are composed of the ER calcium sensor protein STIM and the plasma membrane Orai ion channels ( Table 1 ). Calcium channels like inositol 1,4,5-trisphosphate receptors (IP 3 Rs) and ryanodine receptors (RyRs) in the endoplasmic reticulum release ER Ca 2+ into the cytoplasm, raising intracellular Ca 2+ levels. The elevated intracellular Ca 2+ can further activate calcium release channels. The plasma membrane calcium pump (PMCA) and the sarcoplasmic/endoplasmic reticulum calcium pump (SERCA) regulate Ca 2+ levels by expelling it from the cell or sequestering it into the ER. The sodium-calcium exchanger (NCX) expels Ca 2+ from the cell, while the mitochondrial Ca 2+ uniporter (MCU) absorbs Ca 2+ into the mitochondria, thus maintaining low cytoplasmic calcium levels. We will focus on discussing the regulation of several important channels, transporters, or Ca 2+ ATPases in HNSCC. Section title: VGCCs in HNSCC Educational score: 4.1902689933776855 Domain: biomedical Document type: Study Language: en Recent evidence increasingly highlights the role of VGCC in tumor genesis and progression. VGCCs are classified as low voltage-activated (LVA) or high voltage-activated (HVA) based on their activation threshold. The isomer codes are as follows: L-type (CaV1.1~1.4) encoded by CACNA1S, CACNA1C, CACNA1D, and CACNA1F; R-type (CaV2.3) by CACNA1E; N-type (CaV2.2) by CACNA1B; P/Q-type (CaV2.1) by CACNA1A; and T-type (CaV3.1~3.3) by CACNA1G, CACNA1H, and CACNA1I ( 13 ). Section title: VGCCs in HNSCC Educational score: 4.255000114440918 Domain: biomedical Document type: Study Language: en Previous research suggests that Cav3.1 significantly influences the proliferation and anti-apoptotic activity of human oral squamous cell carcinoma (OSCC) ( 34 ). Cav3.1 expression was overexpressed in OSCC tissues and significantly correlated with Ki-67, PCNA, and Bcl-2 levels ( 14 ). In a study, Cav1.2 was found to be strongly enriched in ameloblastoma (AM) by comparative transcriptome analysis. Cav1.2 primarily mediates Ca 2+ influx in ameloblastoma cells, as demonstrated by the use of agonists and blockers. Genetic study of Cav1.2 demonstrated its direct role in cell proliferation by regulating the nuclear translocation of nuclear factor of activated T cells (NFAT) 1. Cav1.2 is a potential therapeutic target for inhibiting invasiveness during AM progression, necessitating a deeper understanding of the regulatory mechanisms mediating Ca 2+ signaling invasiveness through Cav1.2 ( 35 ). Section title: VGCCs in HNSCC Educational score: 4.367696762084961 Domain: biomedical Document type: Study Language: en α2δ1 is a voltage-gated calcium channel subunit, an essential auxiliary unit of CaV1 and CaV2.In previous studies, it has been demonstrated that α2δ1 plays a crucial role in the regulation of CSC calcium oscillations ( 36 ), and α2δ1 has been reported in both hepatocellular carcinoma and non-small cell lung cancer cells. α2δ1 subunit is a functional marker and a therapeutic target for non-small cell lung cancer and hepatic tumor-initiating cells ( 37 ). A study suggests that α2δ1 has a significant effect on sphere formation or tumorigenicity of laryngeal squamous cell carcinoma (LSCC) both in vivo and in vitro and has the potential to be a tumor stem cells marker for LSCC, and CACNA2D1 is the coding gene of α2δ1 ( 38 ). It was found that overexpression of miR-107 not only reduced the expression level of CACNA2D1 gene, but also inhibited the level of CACNA2D1 protein α2δ1, which could significantly inhibit the malignant biological characteristics of LSCC cells. Thus miR-107 and CACNA2D1 may be potential targets for LSCC prognosis and treatment ( 15 ). Therefore, more studies are needed to fully understand the role of VGCCs in HNSCC. Section title: TRP family channels in HNSCC Educational score: 4.129518508911133 Domain: biomedical Document type: Study Language: en TRP channels, initially identified as a visual mutant in Drosophila, have been demonstrated since the 1990s to exist in various cell line types and healthy human tissues ( 39 , 40 ). Seven subfamilies based on their gene sequence names as follows: ankyrin (TRPA1), canonical (TRPC1 to C7), melastatin (TRPM1 to M8), mucolipin (TRPML1 to ML3), NO-mechano-potential, NOMP (TRPN), polycystic (TRPP2 to P5) and vanilloid (TRPV1 to V6) ( 41 ). TRP channels, primarily situated on the cell surface, engage with various physiological signaling pathways. Gene expression studies indicate that TRP channels play a role in the pathogenesis of HNSCC, with significant implications for diagnosis, prognosis, and treatment ( 6 ). Section title: TRP family channels in HNSCC Educational score: 4.598165512084961 Domain: biomedical Document type: Study Language: en The TRPM subfamily, comprising TRPM1 to TRPM8, is the largest within the TRP superfamily. Multiple studies indicate that TRPM7 is involved in tumorigenesis and various tumor characteristics, including proliferation, migration, invasion, and metastasis. László Köles et al. reported that TRPM7 is expressed in nasopharyngeal, laryngeal, and hypopharyngeal carcinomas ( 42 ). Overexpression of TRPM7 can significantly enhance the migratory ability of non-metastatic nasopharyngeal carcinoma cells. The TRPM7 channel and TRPM7-mediated Ca 2+ influx may be crucial for the migration of nasopharyngeal carcinoma cells ( 43 ). Studies suggest that nasopharyngeal carcinoma patients with TRPM7 overexpression have significantly shorter survival times compared to patients with low TRPM7 expression. TRPM7 is involved in the metastasis of nasopharyngeal carcinoma by enhancing the invasion and migration of nasopharyngeal carcinoma cells ( 16 ). TRPM7 regulates tumor proliferation by continuously activating the JAK2/STAT3 signaling pathway. Compared to high TRPM7 expression, nasopharyngeal carcinoma patients with low TRPM7 expression have higher survival rates. Knocking out the TRPM7 gene can increase the sensitivity of nasopharyngeal carcinoma patients to radiotherapy ( 44 ). TRPM7 is partially involved in the growth and proliferation of human head and neck tumor cell lines ( 45 ). In patients with laryngeal squamous cell carcinoma, circRNAs were first discovered to regulate the expression of TRPM7 ( 17 ). Silencing the expression of TRPM7 can significantly inhibit the metastasis of HNSCC cells, reducing their migration and invasion abilities. TRPM7 is crucial for maintaining HNSCC stem cell characteristics and contributes to chemotherapy resistance. Silencing TRPM7 can inhibit multiple oncogenic signaling pathways and reduce the migration, invasion, colony formation, and tumor sphere formation of human squamous cell carcinoma cells in culture ( 46 ). Research has shown that the anesthetic drug midazolam can inhibit the growth and proliferation of human hypopharyngeal squamous cell carcinoma cells by suppressing the expression of TRPM7 ( 47 ). Section title: TRP family channels in HNSCC Educational score: 4.211306095123291 Domain: biomedical Document type: Study Language: en TRPM2 and TRPM6 are expressed in human oral squamous cell carcinoma ( 18 ). Research indicates that the functions of TRPM2 differ between the membrane and the nucleus. Elevated membrane TRPM2 levels protect against early tumor growth. In the later stages, the loss of membrane TRPM2 and the increase of nuclear TRPM2 enhance the susceptibility to tumorigenesis ( 48 ). Menthol enhances the invasion of oral squamous cell carcinoma cells, while TRPM8 antagonists inhibit this invasion by blocking both menthol-induced and inherent TRPM8 activity ( 49 ). Section title: TRP family channels in HNSCC Educational score: 4.220454216003418 Domain: biomedical Document type: Study Language: en Some studies suggest that TRPV1 is expressed in human OSCC and precancerous lesions. The increased expression of TRPV1 is not related to the malignancy level of the tumor but represents an early step of molecular overexpression in the tumorigenesis process ( 19 ). TRPV1-4 expression was confirmed with higher levels observed in patients with OSCC compared to normal epithelium. Alcohol consumption and smoking are linked to oral cancer and have been shown to elevate TRPV1-4 receptor expression in normal oral mucosa ( 20 ). Capsaicin, a natural TRPV1 agonist, and Capsazepine, a synthetic TRPV1 agonist, are reported to exhibit no cytotoxic effects on non-malignant cells in vitro ( 50 ). Elevated TRPV2 expression correlates with poor prognosis in HNSCC patients, while ANXA6 facilitates autophagy and lymphatic metastasis in HNSCC by modulating TRPV2 expression through mTOR phosphorylation inhibition ( 51 ). Section title: TRP family channels in HNSCC Educational score: 4.225149154663086 Domain: biomedical Document type: Study Language: en Research has found that TRPV4 expression is significantly elevated in nasopharyngeal carcinoma tissues and human nasopharyngeal carcinoma cell lines.TRPV4 stimulates tumor occurrence through Ca 2+ /NFAT4-signaling ( 21 ). Higher expression of TRPV4 is also found in OSCC. TRPV4 induces protein kinase activity that regulates cancer cell growth via activation of AKT ( 22 ). TRPV4 is present in tongue squamous cell carcinoma cells and primary afferent fibers, with cancer pain intensity correlating to the extent of TRPV4 phosphorylation by protease-activated receptor 2 ( 52 ). Section title: TRP family channels in HNSCC Educational score: 4.095381736755371 Domain: biomedical Document type: Study Language: en TRPA1 expression is markedly enhanced in nasopharyngeal carcinoma and OSCC ( 23 ). Notably, OSCC cells can release lipids that activate TRPV1 ( 53 ) and TRPA1 receptors on sensory neurons, leading to pain associated with oral cancer. The up-regulation of TRPA1 protein is associated with the progression of nasopharyngeal carcinoma ( 24 ). Section title: TRP family channels in HNSCC Educational score: 4.236937522888184 Domain: biomedical Document type: Study Language: en Compared with adjacent tissues, the expression of TRPC1 is increased in tongue squamous cell carcinoma tissues, thus having a clinical role in distinguishing and predicting tumor risk ( 25 ). The expression of TRPC1 is positively correlated with the tumor-node-metastasis staging and the depth of invasion in tumor patients. Research indicates that TRPC1 knockdown inactivates the PI3K/AKT pathway, thereby reducing the proliferation and invasion of tongue squamous cell carcinoma cells ( 26 ). The migratory capacity of nasopharyngeal carcinoma cells is associated with TRPC1 expression, and TRPC1 silencing can decrease cancer cell migration ( 54 ). Knock down the expression of TRPC6 located at 11q22 can significantly inhibit the invasion of HNSCC cells. Additionally, amplification and overexpression of TRPC6 have been observed in HNSCC tumor samples ( 55 ). Section title: TRP family channels in HNSCC Educational score: 4.354409217834473 Domain: biomedical Document type: Study Language: en Encoded by the PKD2 gene, TRPP2 is a non-selective cation channel that control calcium signaling ( 56 ). Elevated TRPP2 expression may expedite metastasis in human laryngeal squamous cell carcinoma cells via epithelial-mesenchymal transition (EMT) ( 27 ). Further research indicates that TRPP2 knockout enhances cell proliferation by downregulating the PERK/eIF2α pathway, while the AMPK/ACC pathway activates cell proliferation through feedback mechanisms ( 57 ). The research suggests that cancer cells can effectively absorb exosome/TRPP2 complexes, significantly inhibiting the expression, migration, and invasion of TRPP2 in cancer cells ( 58 ). TRPP2 is also highly expressed in nasopharyngeal carcinoma, promoting its progression by upregulating the Skp2/c-Myc pathway ( 28 ). However, due to the wide tissue distribution and multiple functions of TRP channels, identifying specific TRP channels and their selective cell localization associated with specific cancer-promoting functions is critical for the development of safer and better anticancer drugs. Section title: Store-operated calcium channels in HNSCC Educational score: 4.510072708129883 Domain: biomedical Document type: Study Language: en Store-operated channels are plasma membrane Ca 2+ ion channels regulated by the Ca 2+ content within the endoplasmic reticulum (ER). They are primarily identified as components of a biphasic Ca 2+ signaling mechanism, which includes intracellular Ca 2+ release and Ca 2+ entry via plasma membrane channels ( 59 ). In 1983, it was theoretically proven that receptor-activated Ca 2+ release primarily involves the second messenger inositol 1,4,5-trisphosphate (IP 3 ). A decrease in ER Ca 2+ concentration activates STIM1 and STIM2. The conformational changes in STIM1 and STIM2 stimulate Orai and TRPC channels ( 60 ). The main proteins functioning in CRAC channels have been identified as the ER Ca 2+ sensors STIM and the CRAC channel subunits Orai, which are crucial for the proliferation, migration, metastasis, and apoptosis of cancer cells ( 61 ). Section title: Store-operated calcium channels in HNSCC Educational score: 4.608288288116455 Domain: biomedical Document type: Study Language: en The Orai channel family, comprising Orai1, Orai2, and Orai3, is characterized by significant calcium selectivity. Orai1, the most extensively studied among the three Orai homologs, plays a critical role in cancer progression ( 62 ). Orai1 is a newly identified molecular regulator of carcinogenicity and stemness in OSCC. Orai1 promotes OSCC stemness through the activation of the Ca 2+ -dependent transcription factor NFAT. The Orai1/NFAT pathway regulates hematopoietic stem cells.Orai1 regulates Ca 2+ signaling in OSCC cells and mitigates nociceptive pain and hyperalgesia by controlling collagenase expression among matrix metalloproteinases when deficient ( 63 , 64 ). Additionally, Orai2 is overexpressed in OSCC tissues, significantly contributing to cell proliferation, survival, migration, and metastasis ( 29 ). Orai3 promotes cancer stem-like cell phenotypes by upregulating the stemness transcription factor ID1, suggesting that the Orai3/ID1 axis is a novel regulatory mechanism for maintaining cancer stemness in OSCC ( 30 ). Studies have shown that activation of prostaglandin receptor 4 (EP4) promotes cell migration via PI3K, and the migration of oral cancer cells is regulated by the EP4/PI3K/Orai1 signaling pathway ( 65 ). Studies have shown that not only Orai1 but also STIM1 expression is significantly increased in OSCC ( 31 ). Section title: Store-operated calcium channels in HNSCC Educational score: 4.542385578155518 Domain: biomedical Document type: Study Language: en Studies have shown that HNSCC exhibits overexpression of STIM1 in tumor tissues and is involved in the growth and anti-apoptotic processes of HNSCC, but it is not related to neck lymph node metastasis ( 32 ). Silencing STIM1 inhibits hypopharyngeal carcinoma cell growth and induces cell cycle arrest and apoptosis ( 66 ). Nasopharyngeal carcinoma (NPC), a head and neck malignancy, is linked to the Epstein–Barr virus (EBV) ( 67 ). EBV manipulates the EMT of NPC cells to promote metastatic potential by enhancing STIM1 signaling. Inhibition of STIM1 signaling can suppress the in vivo spread and lymphatic metastasis of NPC cells ( 33 ). Further investigation into the upstream signaling pathways of STIM1 expression indicates that the miRNA-185-5p/STIM1 axis influences the invasiveness of NPC cell lines by modulating cell adhesion through epidermal growth factor receptor (EGFR) activation ( 68 ). EBV enhances EGF-induced STIM1/ERK1/2 signaling. Blocking this signaling pathway may inhibit EBV-enhanced angiogenesis in NPC ( 69 ). Blocking exosome-mediated EBV-associated oncogenic signaling molecules could be an effective strategy for treating NPC ( 70 ). Increasing a long non-coding RNA can inhibit autophagy in cancer cells, and upregulating the PTBP1/STIM1 axis promotes the stemness of nasopharyngeal carcinoma cells ( 71 ). Taken together, the introduction of drugs that specifically target SOCE would be a viable and practical strategy for HNSCC therapy. Section title: Other calcium mediators in HNSCC Educational score: 3.936274290084839 Domain: biomedical Document type: Other Language: en N-methyl-D-aspartate receptors (NMDA) purinergic P2 receptors also regulate Ca 2+ influx. PMCA and NCX mediate the extrusion of Ca 2+ . SERCA pump, IP 3 Rs and RyRs control the movement of Ca 2+ within the ER ( 11 ). Section title: Other calcium mediators in HNSCC Educational score: 4.582890510559082 Domain: biomedical Document type: Study Language: en Research indicates that P2R signaling directly affects pro-inflammatory cytokine production in human OSCC cell lines and promotes cancer progression in healthy host tissues ( 72 ). NMDAR1 is overexpressed in OSCC and is significantly associated with tumor size, lymph node metastasis, and cancer staging ( 73 ). Research demonstrates that salinomycin effectively eliminates cancer stem cells in vivo and in vitro , including in HNSCC cells ( 74 ). Salinomycin’s neurotoxicity is driven by elevated Na + levels, which subsequently increase membrane Ca 2+ via Na + /Ca 2+ exchangers in both the plasma membrane and mitochondria ( 75 ). Inhibiting the mitochondrial Na + /Ca 2+ exchanger can partially mitigate salinomycin-induced neurotoxicity in vivo without compromising its antitumor efficacy ( 76 ). Ca 2+ -ATPases, part of the P-type ATPase superfamily, are categorized into three subtypes based on subcellular localization: plasma membrane Ca 2+ -ATPase (PMCA), ER/SR Ca 2+ -ATPase (SERCA), and Golgi/Golgi-derived vesicles secretory pathway Ca 2+ -ATPase (SPCA) ( 77 ). PMCA1 is abundantly expressed in normal oral epithelial cells but reduced or absent in OSCC cell lines ( 78 ). The reduced expression of ryanodine receptor 2 (RYR2) in tissues adjacent to tumors and in precancerous lesions may be a risk factor for poor prognosis and impending malignant transformation ( 79 ). C17orf104, ITPR3, and DDR2 are among the genes frequently mutated in multiple metastatic or recurrent head and neck squamous cell carcinomas ( 80 ). Section title: Calcium-activated channels in HNSCC Educational score: 4.216259002685547 Domain: biomedical Document type: Study Language: en Calcium-activated potassium (KCa) channels are categorized by their single-channel conductance into large conductance (KCa1.1/BK), intermediate conductance (KCa3.1/IK), and small conductance (SK) channels ( 81 , 82 ). Restoring KCa3.1 activity in HNSCC CD8 + T cells can counteract the inhibitory effects of adenosine. Compared to A2AR receptor inhibition, KCa3.1 activation therapy has greater advantages because it can simultaneously counteract multiple immunosuppressive factors within the tumor microenvironment (TME). Activating KCa3.1 channels may enhance immune surveillance and improve cancer immunotherapy response ( 83 ). Section title: Calcium-activated channels in HNSCC Educational score: 4.443701267242432 Domain: biomedical Document type: Study Language: en Calcium-activated chloride channels (CaCCs) include ANO1 (TMEM16A) and ANO2 (TMEM16B) from the Anoctamin family ( 84 ). The amplification and overexpression of ANO1 and other genes on 11q13 are linked to a higher incidence of future metastases in HPV-negative HNSCC. Functional synergy among these genes may explain their frequent co-amplification at the 11q13 locus ( 85 ). ANO1 is not only a new tumor therapeutic target, but also a predictor of drug therapeutic efficacy ( 86 ). Further studies indicate that TMEM16A is more crucial in HPV-negative HNSCC compared to HPV-positive HNSCC ( 87 ). In HNSCC, the expression of ANO1 is epigenetically regulated through promoter methylation. ANO1 is considered a major driver of the “growth” or “death” pattern in the progression of HNSCC. ANO1 gene amplification frequently occurs in premalignant lesions and invasive tumors ( 88 ). Section title: Drugs targeting Ca 2+ channels/transporters/pumps for cancer treatment Educational score: 3.948288917541504 Domain: biomedical Document type: Review Language: en Mainstream anticancer chemotherapy drugs primarily target DNA replication, DNA/RNA synthesis, DNA damage, and growth factor receptor signaling. Currently, mainstream cancer chemotherapy drugs do not target the Ca 2+ signaling mechanism, possibly because the expression of Ca 2+ channels and transporters on the cell surface can be easily exploited by new drugs or even antibody therapies. Ca 2+ signaling proteins lack specificity, and therapies targeting them might produce unacceptable adverse effects ( 89 ). Recently, novel therapies targeting calcium transporters, channels and pumps are used to treat cancers ( 11 , 90 ), and the related ongoing clinical trials can be observed ( Table 2 ). Section title: TRP channel inhibitors Educational score: 4.242963790893555 Domain: biomedical Document type: Study Language: en The TRPV6 inhibitor SOR-C13 was evaluated in a Phase I clinical trial with 23 patients having advanced solid tumors. Among these patients, one had NPC. The study concluded that SOR-C13 is safe, well-tolerated, and exhibits promising anti-cancer activity, especially in two patients with pancreatic ductal adenocarcinoma ( 91 ). Tranilast is the most extensively studied inhibitor of TRPV2 ( 90 , 97 ). Tranilast has the potential to act as a CAF inhibitor. The inhibition of CAF function by Tranilast can suppress the induction of immunosuppressive cell types in vitro ( 98 ). The text discusses an ongoing study on the use of Tranilast as a radiosensitizer in the reirradiation of NPC. This prospective Phase II interventional trial assesses the safety and efficacy of adding Tranilast for patients with recurrent NPC post-radiotherapy. Studies have found that compared to radiation-sensitive NPC, radiation-resistant cancer tissues are increasingly infiltrated by CAFs. Tranilast treatment demonstrated that CAFs enhance the survival of irradiated NPC cells via the NF-κB pathway, contributing to radiation resistance, which Tranilast can disrupt. This treatment limits the CAF-induced survival of NPC cells and reduces their radiation-resistant characteristics ( 92 ). Section title: T-type calcium channel blockers Educational score: 4.1526594161987305 Domain: biomedical Document type: Study Language: en Blocking T-type Ca 2+ channels can cause cell cycle arrest and reduce cancer cell proliferation. Inhibiting T-type channels alone is unlikely to completely eliminate cancer cells and would need to be combined with standard cytotoxic chemotherapy or radiation therapy ( 99 ). Mibefradil selectively inhibits the T-type calcium channel Cav3, which is primarily involved in calcium influx in various solid cancers, including glioblastoma ( 100 ). Previously used primarily for hypertension and angina, this study is the first to explore Mibefradil as an anti-cancer drug in humans. The study verified that sequentially administering Mibefradil and temozolomide in recurrent high-grade gliomas is safe, and that administering Mibefradil four times daily optimizes systemic exposure to near-maximal drug concentration ( 93 ). This study also has its limitations. In research where overall survival was used as a secondary endpoint, the observed results showed heterogeneity. The study included temozolomide, a drug known to be active against gliomas, making the extent to which Mibefradil enhanced this activity unclear. Additional efficacy trials are required to validate the therapeutic effects of this regimen ( 93 ). ClinicalTrials.gov lists studies on Mibefradil, including NCT01550458, which assesses the safety of administering Mibefradil four times daily in healthy volunteers, and NCT01480050, which explores the combination of Mibefradil Dihydrochloride and Temozolomide for treating recurrent glioma. Section title: SOCE targeted therapy Educational score: 4.108340263366699 Domain: biomedical Document type: Study Language: en Carboxyamido-triazole (CAI), a calcium influx inhibitor, exhibits anti-angiogenic and anti-invasive properties, stabilizing tumor progression ( 101 ). A Phase I clinical trial for recurrent solid tumors established that the maximum tolerated dose is daily CAI administration combined with Paclitaxel injections every three weeks. The study demonstrated that the combination of these two drugs is well-tolerated, active, and potentially effective against several different types of cancer ( 102 ). In the Phase I evaluation of CAI, due to disease stabilization, a Phase II trial was conducted on patients with recurrent ovarian cancer. The trial indicated that CAI shows potential in stabilizing recurrent ovarian cancer with moderate toxicity, warranting further research ( 94 ). Section title: SERCA inhibitors Educational score: 4.209099769592285 Domain: biomedical Document type: Study Language: en SERCA inhibitors, particularly Thapsigargin (Tg), were initially proposed as novel therapeutic agents for cancer treatment and have been extensively studied. Tg induces apoptosis in cancer cells independently of proliferation by inhibiting the SERCA pump ( 95 ). To overcome the high toxicity and non-selectivity of Tg, which disrupts intracellular Ca²⁺ homeostasis in both cancer and normal cells, a Tg-based prodrug strategy has been developed ( 103 ). A prodrug is an inactive compound that can be cleaved by proteases specific to cancer cells ( 104 ). Prostate specific membrane antigen (PSMA) is a carboxypeptidase that is overexpressed in most endothelial cells within prostate cancer and various other tumor types but is not expressed in normal vascular endothelium. G202 is a Tg-based PSMA-activated prodrug targeting PSMA enzyme activity in tumor vasculature, potentially treating various solid tumors ( 105 ). Section title: SERCA inhibitors Educational score: 4.233272552490234 Domain: biomedical Document type: Study Language: en Currently, G202, under the name mipsagargin, has entered clinical trials. Its cytotoxic activity is concealed by a peptide cleaved by PSMA. In a Phase I clinical trial for patients with refractory, advanced solid tumors, mipsagargin exhibited a unique mechanism of action, stabilizing the disease and demonstrating antitumor activity. By using a PSMA-targeted monoclonal antibody combined with the potent microtubule inhibitor MMAE (Monomethyl auristatin E), this approach directly integrates the targeting ability of the antibody with the cytotoxic power of the chemotherapy drug. This provides a precise and effective treatment method with relatively fewer side effects, concentrating the drug’s toxicity on the tumor site and thereby reducing damage to normal cells ( 105 ). Building on promising early clinical trial results, a Phase II clinical trial was conducted in adult patients with hepatocellular carcinoma. The trial established the maximum tolerated dose of mipsagargin as 66.8 mg/m², administered via a 1-hour intravenous infusion on days 1, 2, and 3 of a 28-day cycle ( 106 ). Phase II clinical trials encompassed studies on glioblastoma multiforme , prostate cancer , malignant glioma , and clear cell renal cell carcinoma , among others ( 95 ). Section title: Other drugs Educational score: 4.499565124511719 Domain: biomedical Document type: Study Language: en Calcium electroporation (Ca 2+ -EP) is a novel cancer treatment that benefits the treatment of localized tumors by inducing a significant influx of calcium into cells, leading to cell necrosis ( 107 ). The endoplasmic reticulum, sarcoplasmic reticulum, and mitochondria act as calcium ion reservoirs. Calcium is transported into the ER and SR by the sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA). Elevated intracellular Ca 2+ levels can be toxic, leading to ATP depletion and cell necrosis ( 108 ). Binding ER with Ca 2+ can increase and accelerate the concentration of cellular ions within cancer cells. Calcium electroporation can induce cell necrosis by increasing calcium influx, as demonstrated in vitro on 18 different cell types, showing effects comparable to bleomycin ( 108 ). In vivo experiments on mice with five different human tumors showed varying sensitivity to the treatment ( 109 ). Research indicates that calcium electroporation has anti-vascular effects on both normal and tumor blood vessels in vitro and in vivo , and significantly induces tumor necrosis ( 110 ). Calcium electroporation elicits distinct effects on cell spheroids: it reduces the size of cancer cell spheroids but does not alter the size of normal cell spheroids ( 111 ). Section title: Other drugs Educational score: 4.133495807647705 Domain: biomedical Document type: Study Language: en A phase I clinical trial on calcium electroporation for head and neck tumors included 6 patients, with no observed hypercalcemia, arrhythmia, or serious adverse events post-treatment. One patient achieved complete clinical remission one year post-treatment. Therefore, the future prospects for calcium electroporation are promising, though larger-scale trials are still needed ( 96 ). A phase II study compared calcium electroporation (CaEP) and electrochemotherapy (ECT) for treating skin metastases, including 47 patients, 7 of whom were part of the research protocol. After 6 months of follow-up, calcium electroporation and chemotherapy showed no significant difference in objective response. Seven days after treatment, biopsies collected from tumors treated with either calcium electroporation or electrochemotherapy showed a significant reduction in cancer cell numbers and higher levels of cell death. Compared to electrotherapy, calcium electroporation offers better cosmetic results and is easier to manage due to the lack of cytotoxicity of calcium itself ( 112 ). Section title: Other drugs Educational score: 3.9997007846832275 Domain: biomedical Document type: Study Language: en Extensive research is being conducted on electroporation therapy for head and neck cancer. This nanomedicine and medical technology hold significant clinical potential for cancer treatment ( 108 ). Furthermore, calcium electroporation, which has accessibility and the ability to modulate systemic immune responses, is becoming increasingly popular ( 113 ). Calcium electroporation has been widely used to enhance the treatment of superficial tumors with chemotherapy, and trials for other internal tumors are ongoing ( 114 , 115 ). Calcium is more accessible, easier to manage, and can act as a cost-effective, non-toxic substitute for bleomycin ( 110 ). In a Phase I study for the treatment of keloids, calcium electroporation led to a reduction in thickness by more than 30% in patients with keloids. The treatment was well-tolerated, with no severe adverse reactions or recurrences observed ( 116 ). Calcium electroporation demonstrates therapeutic potential, necessitating further clinical studies to validate its efficacy. Section title: Conclusions Educational score: 4.619281768798828 Domain: biomedical Document type: Review Language: en Calcium signaling is crucial in various cells that facilitate the development and metastasis of HNSCC. Investigating the link between calcium signaling and HNSCC can reveal related genes, pathways, and downstream effectors, emphasizing the clinical importance of altered calcium signaling and identifying new therapeutic targets ( 6 ). This emerging evidence suggests a complex relationship between calcium signaling and the clinical progression of HNSCC ( 11 ). Targeting calcium signaling mechanisms specifically in malignant cells is challenging due to calcium’s widespread role in most cell types and physiological processes. An ideal therapeutic target should be uniquely expressed by cancer cells or exhibit a distinct gain or loss of function to prevent unacceptable adverse effects. Data on calcium signaling mediators’ expression and function in HNSCC patients will be crucial for developing highly effective drugs with minimal side effects ( 11 , 90 ). Despite this, many drugs targeting calcium signaling have been developed, though many are aimed at solid tumors in general rather than specifically at HNSCC. Several of these drugs have demonstrated good safety profiles in Phase I/II clinical trials. Most of these drugs have been assessed in diverse and limited patient populations with various malignancies, with few studies specifically including HNSCC patients ( 89 , 90 ). Calcium channels were expressed in a lot of tissues of the human body, thus, using inhibitors/blockers of calcium channels may cause side effects. Nanomaterial may be a potential strategy for inhibitors/blockers of calcium channels administration to reduce the side effects of targeting calcium channels. Because the nanomaterials can deliver drugs to target tumor cells, which could improve the therapeutic effect by avoiding the delivery of drugs to normal cells. The link between calcium signaling and the reprogramming of cellular energy metabolism remains relatively unexplored. Further research is needed on the possible role of Ca 2+ signaling in glycolytic regulation, glycolytic conversion, and the use of ATP produced by glycolysis to fuel Ca 2+ pumps in cancer cells. The relationship between Ca 2+ signaling and the tumor microenvironment remains unclear. Cancer-associated fibroblasts, for example, are in an “activated” state and interact dynamically with cancer cells, which may be regulated by Ca 2+ signaling. Undoubtedly, calcium signaling mechanisms are an intriguing target for cancer therapy, but the pharmacological opportunities and clinical benefits provided by calcium signaling still need to be further elucidated. | Review | biomedical | en | 0.999997 |
PMC11695300 | Section title: Introduction Educational score: 4.308947563171387 Domain: biomedical Document type: Review Language: en Obstructive sleep apnea (OSA), or obstructive sleep apnea hypopnea syndrome (OSAHS), is a condition in which patients suffer from repeated apnea and hypoventilation during sleep ( 1 ). Common clinical symptoms of OSA can often include loud and irregular snoring, choking or suffocating awakenings at night, sleep disturbances, daytime drowsiness, memory loss, and, in severe cases, cognitive decline and behavioral abnormalities ( 2 ). Researcher widely recognize OSA as a systemic disease that serves as an independent risk indicator for hypertension. It is closely linked to coronary atherosclerotic heart disease (CHD), heart failure, arrhythmias, and diabetes, and is a major cause of sudden death and road traffic accidents ( 3 – 6 ). Inflammation and oxidative stress are the biological mechanisms underlying the relationship between OSA and its various health complications ( 7 ). In the United States, about 26 percent of people aged between 30 and 70 receive an OSA diagnosis, and the prevalence of OSA increased as individual ages increased ( 8 , 9 ). Moreover, eleven population-based epidemiological studies reported 22% OSA in males and 17% OSA in females ( 10 ). As a result, OSA is considered a threat to public health and a serious social problem. Section title: Introduction Educational score: 3.9753220081329346 Domain: biomedical Document type: Study Language: en Medical and epidemiological studies often employ cardiovascular health(CVH) to describe the level of cardiovascular disease risk in an individual or population ( 11 , 12 ). The assessment often considers several risk factors, such as blood pressure, cholesterol levels, diabetes, tobacco use, dietary intake, physical activity, and body mass. As one of the most common respiratory system diseases, OSA, one of the most common respiratory system diseases, may induce hypertension and contribute to CHD, nocturnal angina, myocardial infarction, severe arrhythmias at night, psychiatric abnormalities, respiratory failure, secondary erythrocytosis, and increased blood viscosity ( 13 ). The high prevalence of shared risk factors between OSA and CVH implies a potential synergistic interaction between these diseases, potentially promoting either the initiation or progression of OSA; however, the intricate relationship remains unclear. Therefore, there is an urge to elucidate the intricate relationship of CVH with OSA symptoms. Section title: Introduction Educational score: 3.9564359188079834 Domain: biomedical Document type: Review Language: en Life's Essential 8 (LE 8) is the American Heart Association's(AHA) suggested algorithm to measure CVH, which serves as the vital evaluation for promoting and sustaining cardiovascular health ( 14 ). In 2022, the AHA updated LS7 to Life's Essential 8 to better address the complexities of modern health conditions and the living environment. Numerous studies have extensively studied LS7 metrics and consistently demonstrated an inverse correlation with coronary heart disease(CHD). Recent studies have established a clear inverse relationship between LE8 scores and the risks of cardiovascular disease (CVD) ( 15 ). Section title: Introduction Educational score: 3.6198689937591553 Domain: biomedical Document type: Study Language: en We performed a cross-sectional analysis utilizing data from the National Health and Nutrition Examination Survey (NHANES) to examine the correlation between CVH and OSA symptoms. Our findings suggested a potential association between LE 8 scores and OSA symptoms, even after adjustment for potential confounding factors. Section title: Data source Educational score: 3.4013030529022217 Domain: biomedical Document type: Study Language: en NHANES, overseen by the Centers for Disease Control and Prevention (CDC), surveys the health and nutritional status of adults and children in the US with special emphasis on disease prevalence, adverse risk factors to health, and dietary intake ( 16 ). Public health research and policy is heavily dependent on NHANES data for understanding the overall population health, epidemiology of disease trends, and taking action ( 17 ). All NHANES procedures were conducted per the guidelines of The Ethical Review Committee at the National Centre for Health Statistics, and written informed consent from participants was obtained before initiation. This study adhered to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines for reporting cross-sectional studies ( 18 ). Thus, this study is not required for additional institutional review board approval. Section title: Study design and population Educational score: 4.0334038734436035 Domain: biomedical Document type: Study Language: en In our study, we employed data from NHANES spanning the years 2005–2008 and 2015–2018, comprising a total initial sample size of 39,722 participants. After excluding pregnant women, individuals under 20 years, and those with missing data on OSA symptoms, LE8 score-related indicators, marital status, income level, education level, BMI, alcohol consumption, smoking, and CVD, data from 12,540 participants was included in our analyses . We employ the List-wise Deletion method to remove entire rows that contain any missing values. This approach is advantageous due to its simplicity and ease of implementation, while also preserving the distribution of the data ( Supplementary Material ). Section title: Definition of OSA symptoms Educational score: 3.92478346824646 Domain: biomedical Document type: Study Language: en The assessment of OSA symptoms is based on the responses to three dichotomous questions ( 19 ). The two variables measured in the study were (1) frequency of snoring, (2) frequency of snoring/breathing stops, and (3) frequency of excessive daytime sleepiness. Individuals who exhibited the following characteristics were categorized as having symptoms of OSA: snoring at least 3 times per week, snoring or interrupted breathing at least 3 times per week, and feeling excessively sleepy during the day between 16 and 30 times per month. Section title: Quantification of CVH Educational score: 4.065830707550049 Domain: biomedical Document type: Study Language: en The LE8 metrics is determined by two primary criteria: health behaviors and health factors. It is specifically designed to measure an individual's overall level of CVH. The LE 8 metrics consist of the following components: dietary intake, physical activity, tobacco/nicotine exposure, sleep, BMI, HDL cholesterol, blood glucose, and blood pressure. The study employs an ordinal scoring system with a range of 0 to 100, where lower scores indicate poorer health, while higher scores indicate better health. The total LE8 score for each participant is calculated by summing the scores of all 8 components and dividing by 8. This framework includes comprehensive assessments of CVH at baseline, categorizing scores of <50 for low cardiovascular health; total scores of 50–79 for moderate cardiovascular health, and ≥80 for high cardiovascular health ( 20 – 22 ). Section title: Covariates Educational score: 3.131086587905884 Domain: biomedical Document type: Study Language: en The covariates examined in this study encompass a range of factors, including age, gender (male and female), race and ethnicity (non-Hispanic black, non-Hispanic white, Mexican American, other Latino, and other races-including multiracial), marital status (married/living with a partner, never married, widowed/divorced/separated), personal income ratios [categorized as low income (1.3), middle income (1.3–3.49), and high income (≥3.5)], an education level (<high school, high school, college, or higher), BMI, alcohol, smoking, CVD, diabetes mellitus (DM), hypertension and hyperlipidemia ( 23 – 25 ). Section title: Covariates Educational score: 4.0732421875 Domain: biomedical Document type: Study Language: en We divided the smoking status into three categories: never smokers (less than 100 cigarettes in a lifetime), former smokers (quit after smoking more than 100 cigarettes), and current smokers. We categorized alcohol abuse status into three categories: never drinkers (less than 12 drinks in lifetime), former drinkers (less than 12 drinks in 1 year and no drinks in the last year, or no drinks in the last year but more than 12 drinks in lifetime), and currently drinking. CVD status refers to the existence or nonexistence of any of the subsequent diseases: CHD, congestive heart failure, heart attack, stroke, or angina. The American Diabetes Association criteria and self-report questionnaires define DM, and we consider a case of DM if any of the following conditions applies: (1) Doctor has informed you that you have diabetes; (2) Your glycated hemoglobin HbA1c level is equal to or greater than 6.5%; (3) The fasting blood glucose level should equal or exceed 7.0 mM/L.; (4) The random blood glucose level should equal or exceed 11.1 mM/L. (5) The two-hour OGTT blood glucose level should equal or exceed 11.1 mM/L. (6) Use of diabetes medicine or insulin. Section title: Covariates Educational score: 3.9486677646636963 Domain: biomedical Document type: Study Language: en Hypertension was assessed based on blood pressure measures taken during the physical examination, where systolic blood pressure was equal to or more than 140 mm Hg, or diastolic blood pressure was equal to or greater than 90 mm Hg. Moreover, It also needs to be determined by a physician's diagnosis of hypertension or receipt of hypertension. Definitions of hyperlipidemia include: (1) having a triglyceride level equal to or greater than 150 mg/dl.; (2) increased total cholesterol can be diagnosed if the triglyceride level is equal to or greater than 200 mg/dl, the LDL cholesterol level is equal to or greater than 130 mg/dl, or the HDL cholesterol level is below 40 mg/dl for males or 50 mg/dl for females.; and (3) The use of lipid-lowering medicines is also considered a factor in diagnosing these conditions. Section title: Statistical analyses Educational score: 4.046940803527832 Domain: biomedical Document type: Study Language: en Continuous variables were presented as mean and standard deviation (SD), whereas categorical variables were displayed as frequency and weighted percentage. We used the Chi-square test to assess categorical data and the T -test to compare group differences in continuous variables.Our research aimed to determine the association between CVH and OSA symptoms. CVH is measured by LE 8 metrics (classified into high, medium, and low CVH groups). Participants in the low CVH group served as the reference category. We used a weighted logistic regression model to generate odds ratios (ORs) with 95% confidence intervals (CIs),taking into account into following adjustment variables: age, sex, race, marital status, poverty, education level, BMI, alcohol, smoking, CVD, DM, hyperlipidemia, and hypertension. In Crude model, we did not adjust for any confounders. In model 1, we adjusted for age, sex, and race. In model 3, we additionally adjusted for marital_status, poverty, education_level, BMI, alcohol, and smoking. In model 4, we additionally adjusted for CVD, DM, and hypertension, hyperlipidemia. Section title: Statistical analyses Educational score: 4.034757614135742 Domain: biomedical Document type: Study Language: en In addition, subgroup analyses were performed to investigate if the associations differed by sex (male and female) and age group (<60 and ≥60). Subgroup analyses were performed using stratified logistic regression models. The modifications and interactions of subgroups were inspected by likelihood ratio tests ( 26 ). Each stratification adjusted for the factors (race, marital_status, poverty, education_level, BMI, alcohol, smoking, CVD, DM, hypertension and hyperlipidemia). RCS was employed in weighted logistic regression models to examine the nonlinear relationships between the continuous LE8 and OSAS [Knots = 4, Reference: median (LE8) = 66.25].The selection of the number and location of knots in the RCS was guided by the Akaike information criterion (AIC) to strike a balance between optimal fit and overfitting ( 27 ). Section title: Statistical analyses Educational score: 2.372774362564087 Domain: biomedical Document type: Study Language: en The data were analyzed using R software version 4.2.2 provided by the R Statistical Computing Project and EmpowerStats ( http://www.empowerstats.com ). A two-sided test was applied, and statistical significance was defined as a P value < 0.05.the P value < 0.05 was considered statistically significant. Section title: Population characteristics Educational score: 4.072766304016113 Domain: biomedical Document type: Study Language: en The baseline characteristics of the 12,540 participants in the study sample based on the weighted analyses are presented in Table 1 . The sample comprised 6,360 (51.64%) females and 6,180 (48.36%) males. Among the participants, 3,785 participants (30.18%) had OSA symptoms, whereas 8,755 participants (69.82%) without OSA symptoms. Of the total participants, 8,222 (74.20%) were below 60 years old, and 4,318 (25.80%) were above 60 years old. Mexican Americans accounted for 7.40%, non-Hispanic black people accounted for 9.67%, non-Hispanic white people accounted for 71.77%, and the remaining accounted for other ethnicities, totaling 11.15%. The high CVH group consists of 2,272 participants, of whom 82.79% (1,881) did not exhibit OSA symptoms. In the moderate CVH group, there were 8,687 subjects, with 69.10% (6,003) not exhibiting OSA symptoms. The low CVH group had only 55.09% of participants without OSA symptoms. The mean LE8 score for subjects with OSA symptoms was 63.68, significantly lower than the 70.12 observed in the group without OSA symptoms ( P < 0.001). As depicted in Table 1 participants with OSA symptoms were predominantly male, under 60 years old, married, highly educated, and former or current smokers. In addition, participants with OSA symptoms tend to have a BMI of 30 or higher, reported alcohol consumption, and did not have a history of CVD, DM, hyperlipidemia or hypertension. Section title: Associations of Le8 with OSA Educational score: 4.094878196716309 Domain: biomedical Document type: Study Language: en The results of the sample-weighted multivariate logistics regression analyses are presented in Table 2 . Across all models, there was a significant inverse correlation between symptoms of OSA and LE8 metrics. The 95% CIs for the crude model, model 1, model 2, and model 3 were 0.968 (0.964,0.971), 0.967 (0.963, 0.971), 0.979 (0.974, 0.985), and 0.983 (0.978, 0.988), respectively, all with P < 0.0001. After adjustment for multiple covariates, the 95% CI in model 3 was 0.750 (0.630, 0.893) for the moderate CVH group and 0.573 (0.454,0.723) for the high CVH group. The trend P -value for all models was less than 0.0001, indicating a significant decrease in the risk of developing OSA symptoms with increasing LE8 scores. Section title: Subgroup analyses Educational score: 4.0804243087768555 Domain: biomedical Document type: Study Language: en Subgroup analyses by age and gender in Table 3 specifically examine the relationship between LE8 scores and OSA. The results demonstrate a significant interaction between LE8 scores and OSA symptoms with age . The high cardiovascular health (CVH) group exhibited a lower risk of concurrent OSA symptoms (OR: 0.470; 95% CI: 0.345, 0.641) ( Table 4 ). In the subgroup of male participants, lower LE8 scores were associated with OSA symptoms (OR: 0.982; 95% CI: 0.974, 0.989). In the subgroup of participants aged less than 60 years, lower LE8 scores were significantly associated with OSA symptoms (OR: 0.980; 95% CI: 0.973, 0.986) ( Table 3 ). Section title: Subgroup analyses Educational score: 4.0266876220703125 Domain: biomedical Document type: Study Language: en In summary, for men under 60 years of age, it was shown that those with high CVH grades had a notably reduced risk of experience concomitant OSA symptoms in comparison to those with low CVH grades. There was a notable correlation between the risk ratios and increasing CVH grade, suggesting that CVH grade is a significant risk factor in OSA. A substantial correlation was seen between age and CVH grade, suggesting that age may influence the relationship between CVH grade and OSA. Section title: Dose-response analysis of Le8 score with OSA Educational score: 3.735142469406128 Domain: biomedical Document type: Study Language: en Multivariate corrected RCS analyses indicated a significant inversely nonlinear relationship between LE8 score and OSA . Our finding demonstrates a substantial decrease in OSA symptom prevalence with increased LE 8 scores. Section title: Discussion Educational score: 4.074873447418213 Domain: biomedical Document type: Study Language: en Our study is one of the few to use the NHANES database to conduct a cross-sectional analysis to explore and understand the intrinsic relationship between CVH and OSA. We have observed that CVH subgroups may have a strong association with OSA symptoms, even after adjusting for confounders. The analyses conducted on 12,540 participants revealed that LE8 scores have a negative correlation with the proportion of OSA symptoms. This association was consistent across various demographic characteristics, including gender, race and ethnicity, income, and marital status. However, there was a significant interaction with age. Our findings highlight the importance of maintaining a high CVH to prevent OSA, echoing findings from Zhang et al. ( 28 ). Section title: Discussion Educational score: 4.130393981933594 Domain: biomedical Document type: Study Language: en OSA is a common systemic disease that is closely related to diseases such as CHD and DM, as well as potentially causing sudden death and contributing to road traffic accidents, serving as a serious social problem along with a serious healthcare burden for the public ( 13 , 29 , 30 ). Individuals with OSA experience repeated upper airway obstruction and apnea during sleep, which may lead to a variety of cardiovascular problems, such as decreased oxygen saturation during sleep, a known risk factor for hypertension ( 31 ). Häusler et al. identified an association between sleep-related factors such as OSA and an elevated risk of CVD events, though the exact nature of this relationship remains incompletely understood ( 32 ).Recent research reveals a complex interplay among intermittent hypoxia, oxidative stress, inflammation, and autonomic dysregulation, which collectively contribute to increased cardiovascular risk in patients with OSA ( 33 ). Therefore, given the incomplete investigation of the association and the existing gap in evidence in the US, our cross-sectional study provides essential findings to contribute to the existing knowledge base. Section title: Discussion Educational score: 4.1685028076171875 Domain: biomedical Document type: Study Language: en From the analysis of the 12,540 participants included in the study, we found that the population with concomitant OSA symptoms was characterized by being male, having a BMI ≥30, having an alcohol consumption history, and being a former or current smoker ( 34 ). The prevalence of participants with concomitant OSA symptoms was notably higher among those diagnosed with CVD, hyperlipidemia, hypertension, or DM . This underscores the interconnectedness and shared risk factors among these conditions, such as unhealthy lifestyles, poor dietary habits, obesity, and smoking ( 35 ). We applied the LE8 scoring system to facilitate the quantitative assessment of participants' CVH status. The average LE 8 score was 63.68 in the group with OSA symptoms, which was significantly lower than the 70.12 in group without OSA symptoms . The proportion of participants with concomitant OSA symptoms was considerably higher in low CVH compared to the moderate CVH and high CVH groups. We analyzed the included participants in subgroups by age and gender and found a significant interaction between OSA symptoms with age and LE8 score . In subjects under 60 years of age, a reduced risk of concomitant OSA symptoms was observed in the high CVH group (OR: 0.470; 95% CI: 0.345, 0.641). The OR showed a significant increase with a higher CVH grade, indicating a significant interaction between age and CVH grading. For those under 60 years of age, maintaining a high LE 8 score significantly reduces the probability of developing OSA symptoms. However, this preventive effect appears less pronounced in individuals aged 60 and above. It is widely acknowledged that the risk of OSA may escalate with age, therefore, further research into preventive approaches aimed at mitigating OSA risk among older people becomes vital ( 36 ). Section title: Discussion Educational score: 4.02456521987915 Domain: biomedical Document type: Study Language: en Our study is based on the NHANES database, selecting eligible samples for weighted analysis to explore the association between CVH and OSA. This finding can be extrapolated to the U.S. adult population-offering health policy insights and serving as a foundation for further research in public health interventions. Considering the interrelationship between CVH and OSA, enhancing public awareness and promoting CVH-related diagnosis and treatment may also be an effective means to improve OSA, which serves as an intervention that aims to address potential CVH issues and improve oxygen saturation ( 32 , 37 ). At the same time, maintaining healthy lifestyle habits also serves as an effective method to improve OSA and CVH. Section title: Limitation Educational score: 4.102484703063965 Domain: biomedical Document type: Study Language: en This study also has some limitations. First, the identification of OSA symptoms relied primarily on the typical clinical presentations on the questionnaire, which may not capture all aspects of the condition. Some of the symptoms that are crucial for identifying OSA were not available, such as lifestyle behaviors, which may have resulted in some OSA participants not being identified ( 38 ). Moreover, using questionnaire-based assessments alone may overlook certain cases of OSA. Second, our study is limited to factors such as snoring, intermittent apnea, and drowsiness. This limitation could potentially result in some individuals with OSA not being identified ( 39 ). Third, the participants from the database lacked relevant information about professional laboratory sleep testing, potentially leaving out individuals with asymptomatic OSA from the OSA group. However, these missing individuals are likely to be undifferentiated and have no effect on the final analysis results ( 40 ). Fourth, our study is constrained by its retrospective and cross-sectional design, which introduces information bias stemming from missing data and complicates the establishment of a causal relationship between LE8 scores and OSA. Additionally, we did not employ Directed Acyclic Graphs (DAGs) to identify and elucidate potential confounding variables. Future research will concentrate on this aspect. Section title: Conclusion Educational score: 4.027830123901367 Domain: biomedical Document type: Study Language: en The findings of the research conducted on a large and diverse group of 12,540 individuals in the United States, using data from the NHANES, indicate a strong negative association between LE8 scores and OSA symptoms. Participants with higher LE8 scores were less likely to have concomitant OSA symptoms. These findings underscore the importance of adopting a healthy lifestyle and maintaining a high LE8 score to reduce the incidence of OSA symptoms and their potential role in preventing OSA-related complications.Future research should concentrate on investigating the causal relationships and clarifying the precise mechanisms underlying the relationship between CVH and OSA. | Review | biomedical | en | 0.999997 |
PMC11695308 | Section title: 1 Introduction and aims Educational score: 2.3858442306518555 Domain: biomedical Document type: Other Language: en What does the future hold for two-dimensional (2D) materials? The future is the combination of biotechnology and nanotechnology. The potential applications of nanotechnology in the areas of healthcare and biomedicine are endless. Biological interfacing of 2D materials is a key step towards this paradigm. Section title: 1 Introduction and aims Educational score: 3.2829132080078125 Domain: biomedical Document type: Other Language: en Graphene is the first 2D material isolated, studied and produced of the history, although Sir. Andre Geim acknowledged some early ideas belonging to graphene “pre-history” . Today, after the 20th anniversary of its first characterization, graphene technology is not in its infancy any longer. The impact of research on the innovations, industrialization and commercialization is tangible right now, as proven by the variety of successful patents, commercial spin-offs and start-ups, and products. Some recently published and upcoming ISO standards, are designed to boost the deployment of graphene technology. This is particularly necessary to introduce graphene in the highly regulated markets of products that come in direct contact with human, and to assess the environmental impact of generated waste. Standardized production processes, systematic structural assessment of graphene forms, and specific nomenclature for each member of the graphene family, will in turn facilitate the assessment of structure-properties, structure-safety relationships and market approvals by regulatory agencies. Section title: 1 Introduction and aims Educational score: 4.003327369689941 Domain: biomedical Document type: Review Language: en In this perspective, recent human clinical trials, , intend to unleash a new era of graphene in healthcare applications . In the current landscape of clinical translation, human clinical trials, which ensure rigorous toxicology investigation, performed in highly regulated setting, will build up robust and reliable toxicology data sets as fundaments for future uses in the main themes of biotechnology and medicine: (a) diagnostics, biosensing and imaging; (b) neural interfaces, prosthetic implants and other implantable devices; (c) cancer therapy and drug/gene delivery systems; (d) Tissue engineering and regenerative medicine (albeit this is a cross-functional theme with b and c); (e) antimicrobial and antiviral; (f) industrial bioprocesses, bioreactors and other nano-bio devices. Section title: 1 Introduction and aims Educational score: 1.8704650402069092 Domain: biomedical Document type: Other Language: en This editorial project started in 2019 with Vol I , aims to track advances in the most innovative fields of application of 2D nanomaterials, from graphene to the whole family of novel 2D materials, and their engineered heterostructures, which entails a deep understanding of phenomena happening upon biointerfacing. Section title: 2 Graphene neural interfaces Educational score: 3.6565897464752197 Domain: biomedical Document type: Review Language: en Graphene has proven ideal for neural interface applications, due to its remarkable electrical and mechanical properties, for stimulation and sensing, and high specific surface area for functionalization and cell bio interfacing. Ongoing clinical trials are investigating their functionality and side effects, to assess benefit-risk ratios associated with brain mapping in applications such as cancer brain surgery and treatment of Parkinson disease . Section title: 2 Graphene neural interfaces Educational score: 4.151544570922852 Domain: biomedical Document type: Review Language: en In this article Research Topic, the comprehensive review titled “ Graphene-Based Nanomaterials (GBMs) for Peripheral Nerve Regeneration ” ( Convertino et al. ), considers the exploitation of GBMs biointerfaced, not only with the central nervous system (CNS), but above all with cells associated with peripheral nervous system (PNS), whose ever-increasing attention is being devoted. After describing the development of several graphene production methods, forms (e.g., 3D scaffolds, or conductive coatings) and formulations for nerve repair, the authors critically reviewed graphene interaction with peripheral neurons and, remarkably, highlighted the critical aspect of the local impact of non-neuronal cell alterations induced by the material on nerve regeneration. This no neuro-centric approach is quite broad-minded and highlights the need for a synergic contribution of many cell phenotypes to orchestrate a neuro-regenerative process, laying the foundational evidence of GBM safety and biocompatibility, which is a crucial aspect for clinical translation. Section title: 3 Two-dimensional materials and their multifunctional heterostructures in phototherapy and cancer medicine Educational score: 3.854356050491333 Domain: biomedical Document type: Review Language: en Novel 2D materials, showing a full palette of physicochemical properties, are under the research spotlight. When assembled in Van der Waals vertical heterostructures, nanocomposites or other nanoassemblies not existing in the nature, they display exotic phenomena. Available in different forms such as powders, liquid dispersions, membranes and surface coatings, polymer nanocomposites and hydrogels, foams and aerogels, they virtually find application in any field. Biological interactions with biomolecules and living systems introduce the next functional dimensions. This article Research Topic highlights the significant potential of 2D layered materials and engineered heterostructures for therapy and diagnostics with three articles in the context of phototherapy and cancer theragnostics. Section title: 3 Two-dimensional materials and their multifunctional heterostructures in phototherapy and cancer medicine Educational score: 4.101476192474365 Domain: biomedical Document type: Review Language: en The review by Zhang et al. discusses the unique properties of these materials with recent trends in cancer diagnostics and therapy. The authors emphasize two key aspects. One, is the accurate screening of patients by means of capturing and detection of serum biomarkers, improved MRI/CT imaging capabilities and ultrasound-based diagnostics compared to traditional agents. Two, is the advancement in precision drug delivery including near-infrared radiation-based photothermal (PTT), photodynamic (PTD), sonodynamic, immuno and chemo-therapy. Ultimately, it underscores the promising role of 2D materials in cancer research and treatment, acknowledging the challenges associated with their development. Overall, it emphasizes the differences between 2D materials and other types of traditional nanoparticles (mainly spherical). Section title: 3 Two-dimensional materials and their multifunctional heterostructures in phototherapy and cancer medicine Educational score: 4.139422416687012 Domain: biomedical Document type: Study Language: en Feng et al. reported a groundbreaking photoelectrochemical (PEC) aptasensor, utilizing a double Z-scheme α-Fe 2 O 3 /MoS 2 /Bi 2 S 3 ternary heterojunction, for the ultrasensitive detection of circulating tumor cells (CTCs). A key strength of this work is the successful synthesis of α-Fe 2 O 3 /MoS 2 /Bi 2 S 3 nanocomposite via a step-by-step route. Photoproduced electron/hole transfer path was speculated by trapping experiments of reactive species. The α-Fe 2 O 3 /MoS 2 /Bi 2 S 3 -modified electrodes exhibited greatly enhanced photocurrent under visible light due to the double Z-scheme charge transfer process. All leveraged the unique performances of these materials to achieve high sensitivity and selectivity in CTC detection. The sensor’s broad linear range and low limit of detection are noteworthy, highlighting its applicability in clinical settings. Section title: 3 Two-dimensional materials and their multifunctional heterostructures in phototherapy and cancer medicine Educational score: 4.0455780029296875 Domain: biomedical Document type: Review Language: en The review by Tan et al. present the research progress of 2D materials in Phototherapy . New nanomaterials include nanoelemental nanosheets (Xenes), transition metal carbides, nitrides and carbonitrides (MXenes), transition metal dichalcogenides (TMDs), transition metal oxides (TMOs), layered double hydroxides (LDHs), metal-organic frameworks (MOFs), and Egyptian blue class (XCuSi 4 O 10 ). The authors have meticulously summarized and discussed the recent advances, with several representative examples of anticancer, antibacterial, bone and neural regeneration, cell detection and drug delivery. Section title: 3 Two-dimensional materials and their multifunctional heterostructures in phototherapy and cancer medicine Educational score: 3.2660772800445557 Domain: biomedical Document type: Other Language: en A lesson learned in the Vol II of this article Research Topic is that, whilst graphene has reached a mature stage towards clinical translation, it has also paved the way to a plethora of new 2D materials and heterostructures, which will follow in the upcoming years. Insisting on systematic physicochemical characterization, classification and toxicological evaluation is the key for the deployment of potentially endless technologies enabled by 2D materials and evaluation of risk-benefit ratio in each application. | Other | biomedical | en | 0.999998 |
PMC11695311 | Section title: Introduction Educational score: 3.9886977672576904 Domain: biomedical Document type: Review Language: en Pediatric low-grade gliomas (pLGGs) are the most common brain tumors in children, constituting approximately 30% of all central nervous system (CNS) tumors in this population ( 1 , 2 ). The majority of children with pLGG survive well into adulthood ( 3 , 4 ); this realization has led to a gradual shift in our conceptualization of pLGG into a chronic disease paradigm wherein emphasis is placed on reducing long-term morbidity to optimize functional outcomes ( 5 ). Since the early 1990s, chemotherapy (particularly the combination of carboplatin and vincristine) has been the mainstay of treatment in United States ( 6 ). The 5-year progression-free survival with chemotherapy in patients with sporadic pLGG is around 40% and around 65-85% in patients with neurofibromatosis type 1 (NF-1) associated pLGG ( 7 , 8 ). Though specific chemotherapy protocols vary across the globe, no regimen has shown consistently superior outcomes compared to these historical benchmarks. Section title: Introduction Educational score: 4.171814918518066 Domain: biomedical Document type: Review Language: en Throughout the past decade, multiple landmark studies have provided novel insights into the molecular landscape of pLGGs. The understanding that this disease harbors fewer somatic driver alterations relative to other cancers has made molecularly targeted therapies a particularly exciting avenue. Specifically, the role of mitogen-activated protein kinase (MEK), a serine-threonine kinase in the classic RAS-MAPK signaling cascade, has become a keen area of focus for therapeutic inhibition. In this mini-review, we provide a primer on the role of MEK inhibition in pLGG. The text is organized into the following sections: 1) we recapitulate the key molecular biology mechanisms that underlie pLGG pathogenesis and thereby lay molecular rationale for MEK inhibition as a treatment paradigm, 2) we review the major MEK inhibitors available in the market and present the results of key clinical trials, and 3) we discuss key considerations in the contemporary clinical use of MEK inhibition in pLGG. Section title: Role of MAPK/ERK in pLGG pathogenesis Educational score: 4.632021903991699 Domain: biomedical Document type: Study Language: en Over the last decade, multiple large genomic studies have identified the key somatic driver events for pLGG ( 9 – 13 ). The most important discovery from these studies has been that the disease is driven primarily by alterations in the MAPK/ERK (mitogen-activated protein kinase/extra-cellular signal-regulated kinase) pathway ( 8 ). Canonically, MAPK/ERK is a critical signaling pathway regulating multiple cellular processes including growth, proliferation, differentiation, and survival. In physiological conditions, the pathway is initiated by an extracellular signal (e.g. growth factors, cytokines, hormones) binding to a receptor tyrosine kinase (RTK) which causes dimerization and autophosphorylation. The activated RTK then recruits and activates RAS, a GTPase, which in turn activates RAF, a protein kinase (including the notable isoform BRAF). RAF in turn phosphorylates and activates MEK, another kinase enzyme. MEK activates ERK which translocates into the nucleus where it phosphorylates transcription factors thereby altering gene expression. Relatedly, neurofibromin (a protein encoded by the NF1 gene) accelerates deactivation of RAS thus acting as a negative regulator of the pathway . Section title: Role of MAPK/ERK in pLGG pathogenesis Educational score: 4.663129806518555 Domain: biomedical Document type: Study Language: en Structural variations in the BRAF gene are the most common somatic driver event for sporadic (non-NF1-related) pLGG, found in roughly 70% of cases ( 13 , 14 ). These fusion events create abnormal gene products such as KIAA1549-BRAF which lead to the constitutive activation of the MAPK pathway. A smaller, but still significant, subset of pLGGs harbor single nucleotide polymorphisms in BRAF (most frequently BRAF V600E ) which result in upregulated protein activity ( 15 ). One in five children with NF1 develop pLGG in the central nervous system, with approximately one third occurring in the optic pathway ( 16 ). Over 90% of pLGGs in NF1 patients harbor bi-allelic inactivation of NF1 without the BRAF mutations that are common in sporadic cases ( 17 ). A subset of NF1-assocaited pLGGs carry a higher risk of progression and treatment-resistance. This clinical heterogeneity is thought to be driven by acquisition of additional molecular alterations ( 18 ). For example, alterations in ATRX, CDK2A, and TP53, genes typically associated with high grade gliomas, are found infrequently in NF1-associated pLGGs and may portend more aggressive behavior. Section title: Rationale for MEK inhibition Educational score: 4.333423137664795 Domain: biomedical Document type: Study Language: en Given the central role of the MAPK pathway in pLGG pathogenesis, targeting MEK, a key kinase in this pathway, presents a rational therapeutic strategy. MEK inhibitors can potentially interrupt the aberrant signaling cascade driving pLGG pathogenesis, thereby halting tumor progression and inducing tumor regression in some cases. MEK inhibition is promising because of the potential for target specificity in comparison with classic chemotherapy (carboplatin and vincristine) raising the possibility of more limited toxicities and side effects. Furthermore, there is a key distinction between MEK1/2 and other protein targets within the MAPK/ERK pathway; MEK1/2 are kinases that activate ERK1 and ERK 2 but do not have any other known physiological substrates ( 19 ). In contrast, ERK1/2 catalyzes phosphorylation of countless cytoplasmic and nuclear targets for a diverse array of cellular functions. MEK1/2 thus serves as a relatively selective (and thus strategic) targetable bottleneck in tumorigenesis. Section title: Rationale for MEK inhibition Educational score: 4.089990139007568 Domain: biomedical Document type: Study Language: en Though we will focus here on the role of MEK inhibition, it is worthwhile to note that in 2024 the FDA approved a highly selective type II RAF kinase inhibitor (Tovorafenib) pursuant to the results of a multicenter, open-label, single-arm trial in children with relapsed or refractory pLGG harboring an activating BRAF alteration ( 20 ). The approval of Tovorafenib serves as an encouraging proof of concept supporting a focus on highly selective targeting of the RAS/RAF/MAPK pathway in pLGG. Relatedly, LOGGIC/FIREFELY-2 is currently enrolling patients and will serve as a Phase 3 trial investigating the efficacy of Tovorafenib monotherapy compared with chemotherapy in children with newly diagnosed LGG harboring an activating RAF mutation ( 21 ). Section title: Major MEK inhibitors that have been clinically adopted Educational score: 3.744208335876465 Domain: biomedical Document type: Review Language: en Multiple trials have experimented with the use of MEK inhibitors in different cancers that harbor mutation in the MEK pathway. Here, we review the available MEK inhibitors and the published clinical data on their role in pLGG ( Table 1 ): Section title: Selumetinib Educational score: 4.212104797363281 Domain: biomedical Document type: Study Language: en Selumetinib is an orally available non-ATP-competitive small molecular inhibitor of MEK1/2. In 2017, a phase I trial of selumetinib in pediatric patients with recurrent or refractory pLGG was published by the Pediatric Brain Tumor Consortium (PBTC). The trial established a recommended phase 2 dose (RP2D) and reported a 2-year progression-free survival (PFS) at RP2D of 69% (SE 9.8%) ( 22 ). In a subsequent phase II trial in children with BRAF-aberrant or NF1-associated recurrent, refractory, or progressive LGG, imaging response rates were in the 30-40% range ( 23 ). Of the patients with BRAF aberrations (n=25), 36% achieved sustained partial response (PR) (median follow-up 36.4 months). Similarly, patients with NF1-associated tumors (n=25) achieved 40% sustained PR (medial follow-up 48.6 months). The study also reported on visual outcomes in 10 patients with optic pathway gliomas (all NF1-associated); 8 reported stable vision and 2 reported improvement. Elevated creatinine phosphokinase and maculopapular rash were the most frequent grade 3 or higher adverse events, both occurring in about 10% of patients. Notably, 36% of patients required a dose reduction due to toxicity, either grade 2 or grade 3. Although common, elevation of creatine phosphokinase was not symptomatic in most treated patients and the requirement for dose reduction based solely on this laboratory finding is an ongoing discussion in the treating community. The results of this phase II trial generated great excitement given that the tumor response with selumetinib therapy appeared comparable to that with chemotherapy but with significant advantages including oral administration without the need for central lines, no immunosuppression, less hair loss, and reduction in number of clinic visits ( 24 ). Results in a separate stratum of the same phase II trial that involved recurrent optic pathway or hypothalamic LGG in patients without NF1 (n=25) showed that 24% had PR, 56% had stable disease (SD), and 20% had disease progression ( 25 ). Visual acuity improved in 26% and was stable in the rest. Section title: Selumetinib Educational score: 3.697457790374756 Domain: biomedical Document type: Other Language: en These results led to the current phase III, non-inferiority trials through the Children’s Oncology Group (COG) comparing selumetinib with standard-of-care chemotherapy (carboplatin and vincristine) for newly diagnosed pLGG. ACNS1821 is being conducted in children with NF1-associated pLGG, while ACNS1833 is being conducted in children with non-NF1 and non-BRAF V600E mutant pLGG. Section title: Selumetinib Educational score: 4.087973594665527 Domain: biomedical Document type: Study Language: en Though not in the context of pLGG, we note that compelling long term safety data for the use of selumetinib comes from the SPRINT trial conducted in the context of inoperable symptomatic plexiform neurofibromas (PN) in children with NF1. In the phase I study (n=24), investigators had reported only 1 grade 4 AE (asymptomatic elevated CPK). In the phase II study (n=50), three grade 4 AEs had been reported (CPK increase, hyperuricemia, and skin ulceration). In a five-year follow-up report after publication of phase II results, investigators reported that there had been no new or concerning safety signals in patients from the phase I or phase II studies ( 26 ). Section title: Trametinib + Dabrafenib Educational score: 4.228346824645996 Domain: biomedical Document type: Study Language: en Trametinib is a reversible, orally bioavailable, allosteric inhibitor of MEK1/2. In 2022, the FDA approved the combination of trametinib and dabrafenib (a BRAF inhibitor) in a tumor-agnostic fashion for the treatment of patients ≥ 6 years of age with unresectable/metastatic BRAF V600E-mutant solid tumors that have progressed after prior treatment and have no satisfactory alternative. As part of the supporting data for this approval, Bouffet et al. reported initial results from NCT02124772, a phase I/II study in children with relapsed/refractory BRAF V600-mutant pLGG treated with either trametinib monotherapy or in combination with dabrafenib ( 27 ). The PR rate was 15% in the monotherapy group (n=13) and 25% in the combination therapy group (n=36). Median PFS with combination therapy was 36.9 months, compared to 16.4 months with monotherapy. In the monotherapy group, common adverse events were paronychia (54%), diarrhea (46%) and dry skin (46%). Adverse events led to discontinuation in 54% of patients. In the combination group, the most common adverse events were pyrexia (50%) and dry skin (42%). Adverse events led to discontinuation of combination therapy in 22% of patients. A follow-up phase II trial, NCT02684058, studied the use of combination trametinib + dabrafenib (T+D) as first-line treatment for BRAF V600-mutant pLGG compared with chemotherapy (C+V) ( 28 ). Seventy-three children were treated with MAPK inhibition (T+D) and 37 with chemotherapy (C+V). The trial reported a higher overall response rate (ORR) in the group treated with MAPK inhibition (47% vs 11%, p< 0.001) and higher median PFS (20.1 months versus 7.4 months, p<0.001) when compared to the chemotherapy group. The most frequent adverse events in the T+D group were pyrexia (68%), headache (47%), and vomiting (34%), but there were fewer grade ≥3 adverse events in the T+D group and fewer discontinuations (4% versus 18%) as opposed to the carboplatin and vincristine group. Subsequent to the emergence of these data, the FDA approved D+T for children ≥1 year of age with newly diagnosed BRAF V600E mutant pLGG in March 2023. Given these data, the combination of trametinib and dabrafenib is now considered the standard of care for this select group of patients. Section title: Trametinib + Dabrafenib Educational score: 3.9606144428253174 Domain: biomedical Document type: Study Language: en The utility of trametinib as a single agent in pLGGs without BRAFV600E alterations has not been well studied and currently only retrospective cohort data are available ( 29 – 31 ). Manoharan et al. reported on a retrospective cohort of patients treated with trametinib for progressive pLGGs and demonstrated a small percentage of PR and minor responses (MR) with most patients having SD on therapy ( 31 ). Section title: Trametinib + Dabrafenib Educational score: 3.9642868041992188 Domain: biomedical Document type: Study Language: en Additional studies focused on trametinib monotherapy are currently underway, including PLGG-MEKTRIC, a large prospective trial comparing daily trametinib monotherapy with weekly vinblastine in children with non-NF1 associated pLGG and without BRAFV600E mutation in France , and TRAM-01, a phase 2 open-label basket trial in Canada , is testing trametinib as a single agent in pediatric patients with progressing/refractory glioma or plexiform neurofibroma with MAPK.ERK pathway activation. Section title: Binimetinib Educational score: 4.012336730957031 Domain: biomedical Document type: Other Language: en Binimetinib is an oral selective MEK 1/2 inhibitor currently FDA approved in the US for use in combination with BRAF inhibitor encorafenib in adult patients with unresectable BRAFV600 altered melanoma and non-small cell lung cancer. A multi-institutional phase II trial was conducted to evaluate the use of binimetinib in children with previously treated radiographically progressive pLGG ( 32 ). Radiographic response rates at 1 year were: 50% in pLGG with BRAF mutation (total n=28, 12 PR, 2 MR), 43% in NF1-associated pLGG (total n=21, 5 PR, 4 MR), and 69% in sporadic pLGG without BRAF mutation (total n = 29, 10 PR, 10 MR). The most common toxicities, as with other MEK inhibitors, were predominantly dermatologic and therapy had to be discontinued in 22% of patients, while 49% required a dose reduction due to toxicity. Section title: Cobimetinib Educational score: 4.03978157043457 Domain: biomedical Document type: Study Language: en Cobimetinib is an orally active, highly selective small molecule inhibitor of MEK1. iMATRIX-cobi was a multicenter phase I/II study investigating cobimetinib in pediatric patients with relapsed or refractory solid tumors with known/expected MAPK pathway involvement ( 33 ). Thirty-two patients with LGG were enrolled. Three patients (9%) had PR and 18 (56%) had SD (median follow-up 15 months). Eleven percent of patients (n=6) experienced adverse events requiring discontinuation from drug. It should be noted that 5 out of 6 of these patients experienced an ocular toxicity: one patient had grade 2 retinal detachment, 2 had chorioretinopathy (1 grade 2, 1 grade 4), one had grade 1 pigment epithelial detachment, and one had grade 1 serous retinal detachment. The higher incidence of ocular toxicities with this agent is notable given the interest in using MEKi for optic pathway gliomas wherein the tolerance for such adverse events is particularly low. Section title: Mirdametinib Educational score: 3.916367769241333 Domain: biomedical Document type: Other Language: en Mirdametinib is an oral small molecule allosteric inhibitor of MEK1/2. Though not yet available in the market, a New Drug Application (NDA) for the agent has recently been accepted by the FDA. SJ901 is a multi-arm phase I/II trial of mirdametinib in recurrent/progressive MEKi-naïve pLGG (excluding BRAFV600); results from phase I have recently been published ( 34 ). In 23 enrolled patients with median follow-up of 14.6 months, only one dose limiting toxicity (grade 3 thrombocytopenia) was seen. Twelve (63%) of the nineteen patients with measurable tumors achieved and objective response (OR) (of these, 1 major, 6 partial, and 5 minor). RP2D has been established (3mg/m 2 /dose BID) and phase II is ongoing. Section title: Patient selection Educational score: 4.2045440673828125 Domain: biomedical Document type: Study Language: en Determining appropriate selection of patients is perhaps the most challenging aspect of translating our burgeoning understanding of the molecular basis of pLGG into clinical practice. Not all pLGG – even with very similar genetic profiles - are likely to benefit equally from MEK inhibition and evidence-based decision-making paradigms are scant. Sigaud et al. have recently published results for a MAPKi sensitivity score (MSS) ( 35 ). Using pLGG transcriptomic data from large databases, they were able to show that computational models could predict the response of cell lines to drugs inhibiting different aspects of the MAPK pathway including MEKi. A key finding of their work was that accurate predictions relied not only on genetic information from the tumor cells but also the tumor immune microenvironment. Higher susceptibility scores correlated with higher immune cell infiltration (i.e. high expression in microglia compartment in single-cell RNA sequencing). This latter finding underscores the enormous complexity involved in predicting how a given patient will respond to MAPK inhibition based on molecular markers alone. Though not yet prospectively validated and thus not currently generalizable to routine clinical practice, the Sigaud method allows us to be optimistic about the development of computational tools that could be used in concert with the genetic profiling which is increasingly routine in the care of pLGG patients to make more personalized data-driven treatment recommendations. Section title: Treatment duration and post-discontinuation re-growth Educational score: 4.115273475646973 Domain: biomedical Document type: Study Language: en There is no consensus on the appropriate duration of treatment with MEK inhibitors for pLGG, though 2 years has become a common yardstick in clinical practice as this was the duration of most pLGG focused trials(though without a clear scientific rationale) ( 36 ). Use of MEK inhibitors is not limited to the pediatric population, however, and their use has been longstanding in adult tumors, particularly in melanoma. An increasingly common paradigm is to cease therapy after reaching prolonged CR. This notion is based on small cohort studies wherein most patients who achieved prolonged CR were monitored long term (8-36 months) without recurrence ( 37 ). It is challenging, however, to translate this principle into the care of pLGG patients treated with MEKi as CR is rare in our patients. Differences in tumor biology and behavior require adjustments to treatment strategy. Some pLGGs will remain stable for many years in a state of clinical senescence. Prolonged exposure to MEKi and the potential toxicities may not be warranted for this length of time and is likely not sustainable for most patients. Furthermore, the phenomenon of clinical senescence greatly complicates our ability to confidently ascertain whether tumor stability is related to therapeutic MEK inhibition as opposed to the natural history of the tumor. Section title: Treatment duration and post-discontinuation re-growth Educational score: 4.104788780212402 Domain: biomedical Document type: Study Language: en A related concern in pLGG, not seen in most adult indications, is the rapid regrowth of disease soon after discontinuation. In the phase II selumetanib trial discussed earlier, 56% of patients with sporadic pLGG had disease progression with the majority occurring after therapy discontinuation. More strikingly still, in the NF1-associated pLGG stratum (n=25), only one patient progressed while on therapy while seven progressed after therapy discontinuation ( 23 , 24 ). These studies have raised concern about the need for prolonged therapy in a subset of patients with recurrent, progressive disease and the possible merits of a slow wean of the drug. Such a ‘slow wean’ approach for MEKi has recently been endorsed in a consensus statement by Canadian experts ( 38 ). Section title: Retreatment Educational score: 4.125790119171143 Domain: biomedical Document type: Study Language: en Retreatment of patients with the same or alternative MEK inhibitors after progression with discontinuation of drug has become more accepted in clinical practice. There is some initial evidence supporting this type of re-retreatment. In the adult melanoma literature for instance, retreatment with combination MEK and BRAF inhibition is reasonable and is associated with at least temporary response ( 39 ). Specifically, in pLGG, preliminary evidence suggests that retreatment with selumetinib in patients who progress after initial therapy may be effective in restoring response or stability ( 40 , 41 ). A key area of ongoing investigation is whether the addition of anti-resistance agents to MAPKi regimens may be a useful adjunct in retreatment paradigms. A current phase I/II trial through the PBTC is testing addition of hydroxychloroquine to the existing combination of trametinib and dabrafenib (T + D + HCQ) in patients previously treated with a RAF or MEK inhibitor. The addition of HCQ is based on preclinical evidence showing that the agent can inhibit treatment induced autophagy which is a known pathway to treatment resistance in pediatric gliomas ( 42 ). Safety data for 18 evaluable subjects enrolled in the phase I component were comparable to prior MEK inhibitor trials; there were 2 dose-limiting toxicities, both grade 3 rash, and the highest dose level of HCQ was declared the RP2D ( 43 ). Section title: Intermittent dosing Educational score: 4.18447732925415 Domain: biomedical Document type: Study Language: en There has been substantial interest in the idea of using intermittent dosing (or ‘drug holidays’) as a way of mitigating treatment related toxicities while potentially decreasing resistance to MAPK inhibition. Again, population specific evidence for pLGG is lacking but reports from adult studies may be at least partially informative. A randomized phase II trial in adult melanoma patients investigated continuous versus intermittent dosing of combination trametinib and dabrafenib in metastatic or unresectable BRAF V600E - mutant melanoma ( 44 ). There were no significant differences in terms of toxicity or OS however patients in the continuous dosing arm had improved PFS. In another trial, continuous dosing of combination cobimetinib and vemurafenib (a Type 1 BRAF inhibitor) was compared with intermittent dosing in patients with advanced BRAF-mutant melanoma ( 45 ). Again, patients in the intermittent dosing arm did not have improved PFS or substantially reduced toxicities. Though these adult melanoma studies are imperfect proxies for the pLGG context given the significant difference in the disease pathophysiology and natural history, they do provide evidence for the notion that an intermittent dosing approach should be viewed with caution at this time. Clinical trials testing the specific question of whether intermittent dosing may be beneficial for pediatric glioma patients are underway; NCT03326388 (INSPECT) is a phase I/II study in children with NF1-associated tumors (inoperable plexiform neurofibromas and recurrent optic pathway gliomas) being treated with intermittent dosing of selumetinib. Notably, the recently published ReNeu trial found that children with NF-1 associated inoperable symptomatic plexiform neurofibromas treated with intermittent dosing of Mirdametinib (3 weeks on/1 week off) met the primary efficacy endpoint, with ORR of 52% ( 46 ). Section title: Potential impact on fertility Educational score: 4.008020401000977 Domain: biomedical Document type: Study Language: en Given the long term survival of most pLGG patients, there has been a sustained interest in establishing the impact of targeted therapies (including MEKi) on fertility, but compelling data in human subjects remain scant. Concerns about the potential toxicity to fertility come from animal studies. In female rats, cobimetinib exposure increases apoptosis and necrosis of cells in the corpus luteum and vaginal epithelium while also causing testicular degeneration in male dogs ( 47 ). Trametinib has also been associated with reduced corpus leuteum cells in female rats, but no observed effect in male reproductive tissues ( 47 ). To our knowledge, there are currently no data in humans to better inform the long-term risks to fertility associated with MEKi use ( 48 ). Section title: Discussion Educational score: 4.323101043701172 Domain: biomedical Document type: Review Language: en While chemotherapeutics have been the mainstay for the treatment of pLGG since the early 1990s, our understanding of the molecular drivers of the disease has deepened markedly in the last decade leading to the widespread appreciation of the role of targeted therapies. Unlike other cancers, pLGG is driven by relatively few somatic driver mutations primarily impacting the MAPK/ERK pathway. MEK inhibition has now been evaluated in multiple phase I/II trials in both sporadic and NF1 associated pLGG and early results suggest that for appropriately selected patients outcomes are likely to be as good as (if not superior to) those with conventional chemotherapies. The emergence of the MAPK pathway alterations and the activity of targeted agents has made molecular characterization of the tumor a required aspect of diagnosis and management. Several questions remain, however, in the continued use of this targeted therapy. Identification of the patients most likely to benefit, the durability of therapy, the length of optimal therapy, and the process for therapy discontinuation remain clinical challenges. Much work is necessary to resolve these challenges before the treatment paradigm can shift to one wherein molecular targeted therapies are the standard treatment. Combination of MEKi with cytotoxic chemotherapy or combination of MEKi with other targeted therapies such as PD1 inhibition may be exciting future avenues that are yet to be explored. | Review | biomedical | en | 0.999995 |
PMC11695313 | Section title: 1 Introduction Educational score: 4.006853103637695 Domain: biomedical Document type: Review Language: en Uterine fibroids are the most prevalent benign tumors of the female reproductive system, characterized by high vascularity, with incidence rates increasing with age. The symptoms associated with fibroids can appreciably impair women’s quality of life . Traditional treatment modalities primarily encompass hysterectomy, myomectomy, laparoscopic myomectomy, and uterine artery embolization . Recently, non-invasive high-intensity focused ultrasound (HIFU) has gained prominence in the management of symptomatic uterine fibroids and is now included in treatment guidelines in several countries, with its effectiveness extensively corroborated . Nevertheless, due to the inherent properties of fibroid tissue and technical constraints, HIFU may not be suitable for all patients . Therefore, precise preoperative evaluation is imperative for the successful application of HIFU. Enhancing the accuracy of preoperative predictions regarding HIFU treatment efficacy is crucial for clinicians to formulate optimal treatment strategies. Section title: 1 Introduction Educational score: 4.407512664794922 Domain: biomedical Document type: Review Language: en Preoperative magnetic resonance imaging (MRI) with HIFU is instrumental in the differential diagnosis of uterine fibroids, as well as in assessing their location, morphology, and potential tissue composition . MRI is recommended as an essential preoperative evaluation tool for HIFU treatment of uterine fibroids. The imaging characteristics of fibroids across various MRI sequences exhibit a notable correlation with their tissue heterogeneity. Pervious research has elucidated that T2-weighted imaging (T2WI) offers superior visualization of the internal architecture and morphology of uterine fibroids, facilitating the differentiation of cellular hydration levels from fibrous tissue composition . Moreover, contrast-enhanced T1-weighted imaging (CE-T1WI), achieved through the administration of contrast agents, augments the delineation of fibroid boundaries relative to the surrounding tissues, thereby enhancing the assessment of vascularization . Consequently, it assists in evaluating the proliferative activity and invasive potential of the fibroids. These imaging characteristics are instrumental in enabling clinicians to subjectively assess the tissue characteristics of fibroids and forecast the efficacy of ultrasound energy in inducing coagulative necrosis in the targeted region. Despite these advances, challenges remain due to the variability in clinical experience and the inherent limitations of human visual assessment in accurately evaluating fibroid morphology, spatial distribution, and lesion characteristics . Section title: 1 Introduction Educational score: 4.529837131500244 Domain: biomedical Document type: Study Language: en Radiomics is revolutionizing medical imaging by enabling the detailed analysis of tumor complexity at a microscopic level through technological advancements . By utilizing high-throughput imaging data, radiomics overcomes the limitations of traditional imaging, revealing subtle features that are often imperceptible to the naked eye . This cutting-edge technology not only enhances the precision of tumor diagnosis but also facilitates personalized treatment, providing unparalleled insight into the potential threats posed by tumors. In the preoperative assessment of HIFU ablation of uterine fibroids, radiomics models have demonstrated significant clinical potential. However, previous studies have primarily relied on radiomics features from single MRI sequences for efficacy prediction . While some progress has been achieved, single sequences often fail to comprehensively represent the biological information of fibroids, leading to limited predictive accuracy. By integrating information from different MRI sequences, a more comprehensive understanding of the tissue characteristics and biological properties of fibroids can be achived, which significantly enhancing the accuracy and stability of prediction models . Nonetheless, existing research has not fully incorporated the CE-T1WI sequence, which most accurately reflects fibroid blood supply, and has overlooked the importance of including diverse MRI sequences in the model to enhance its performance. To facilitate this integration, deep learning (DL) based automatic segmentation plays a crucial role by providing precise delineation of fibroid boundaries and relevant anatomical structures across multiple MRI sequences . As Imran Iqbal demonstrated, deep learning can effectively extract disease-related information and address challenges such as limited annotated data by leveraging pre-trained models, which has proven beneficial in detecting various medical conditions, including joint abnormalities and skin cancer screening . Such methodologies not only enhance the accuracy of segmentation but also provide a solid foundation for the subsequent extraction of radiomic features, ensuring both the efficiency and consistency of the analysis. The application of automated segmentation techniques allows researchers to rapidly process large volumes of imaging data, thereby improving the reliability and predictive power of model construction. However, in the realm of machine learning applications, traditional single algorithms and hyperparameter tuning methods, although effective, may not fully exploit the potential of the radiomics features. Stacking, as an advanced ensemble learning method, demonstrates substantial potential in enhancing predictive performance . Stacking first trains multiple types of base learners to capture diverse features and patterns within the data. Subsequently, the outputs from these base learners are used as inputs to train a meta-learner, which integrates the strengths of the base learners and addresses their shortcomings, ultimately producing more accurate results. This method leverages the advantages of multiple models to effectively handle complex data, reduce overfitting, and improve prediction accuracy and stability . This study aims to develop a multimodal MRI radiomics analysis method that integrates automated segmentation and stacking ensemble learning techniques to further enhance the predictive performance of HIFU ablation of uterine fibroids. Section title: 2.1 Patients Educational score: 4.192049503326416 Domain: biomedical Document type: Study Language: en We conducted a retrospective analysis involving 1457 consecutive patients diagnosed with uterine fibroids across two medical centers. To accurately assess the efficacy of HIFU ablation for uterine fibroids while controlling for various factors on efficacy, such as fibroid size, location, tissue composition, abdominal wall thickness, and the presence of abdominal wall scars. We established the following inclusion criteria : patients over 18 years old ; no prior surgical interventions or relevant medication history ; fibroids located in the anterior uterine position ; fibroids measuring between 3–8 cm in diameter ; abdominal fat thickness ranging from 1 to 3 cm; and for patients with multiple fibroids, only the largest fibroid was included. The exclusion criteria were : history of pelvic surgery or other concurrent tumors; and imaging artifacts that interfered with accurate assessment. A non-perfused volume ratio (NPVR) ≥80% was used to define treatment efficacy, a threshold validated across different levels of physician expertise . Thus, an NPVR of ≥80% was considered indicative of a favorable prognosis, while an NPVR <80% was deemed an unfavorable prognosis. NPVR assessments were independently performed by two radiologists: one with 4 years and the other with 15 years of diagnostic experience. Discrepancies were resolved in favor of the more experienced radiologist. After a stringent screening process, we included 300 patients from Center A and 60 from Center B, dividing them into three groups: a training set (N = 240), an internal test set (N = 60), and an external test set (N = 60). Approval for the study was granted by the Institutional Review Boards of both centers , and the need for written informed consent was waived. The patient enrollment process is detailed in Figure 1 . Section title: 2.2 Images acquisition Educational score: 3.625333547592163 Domain: biomedical Document type: Study Language: en MRI scans were acquired from two centers: one using a 3.0 T Signa HDxt MRI scanner and the other using a 1.5 T Signa Voyager MRI scanner, both provided by General Electric. Patients were positioned in the supine position and scanned using a dedicated 8-channel phased array coil for the abdomen. Detailed MRI acquisition parameters are shown in Table 1 . Section title: 2.3 Image segmentation and feature extraction Educational score: 4.122457504272461 Domain: biomedical Document type: Study Language: en This study employed a V-Net architecture for automatic segmentation of pelvic MRI data, specifically targeting uterine fibroids in T2WI and CE-T1WI . The network is based on an encoder-decoder architecture. The encoder uses 3D convolutional modules to extract features from medical images and adjusts the feature resolution through convolution operations with a stride of 2. In the decoder, 3D transposed convolutions are used to progressively restore the deep semantic features extracted by the encoder to a higher resolution. Skip connections are incorporated between the encoder and decoder to effectively combine low-level and high-level features, thereby enhancing segmentation accuracy. The network includes three auxiliary loss layers and one main loss layer; the main loss layer employs 3D transposed convolutions to recover the feature maps to the original image size, ultimately outputting the automatically segmented target regions. Figure 2 illustrates the architecture of the automatic segmentation network, while Figure 3 presents the segmentation results. We evaluated the model’s performance on the validation data set, which yielded an average Dice Similarity Coefficient (DSC) of 0.883 for the T2WI segmentation model and 0.809 for the CE-T1WI segmentation model, indicating good performance in the automatic segmentation for uterine fibroids. Section title: 2.3 Image segmentation and feature extraction Educational score: 4.110904693603516 Domain: biomedical Document type: Study Language: en After the automatic segmentation of the remaining MRI scans, two independent radiologists evaluated the segmented regions. For images with inaccurate outlines, manual corrections were made using ITK-SNAP software (version 3.8, http://www.itksnap.org ) to create complete Regions of Interest (ROIs). Prior to feature extraction, all MRI images and segmentations were resampled to a voxel size of 1 × 1 × 1 mm 3 using bilinear interpolation. Features were extracted from the T2WI and CE-T1WI ROIs using Python (version 3.10, https://www.python.org ), focusing on both low-dimensional and high-dimensional aspects. Low-dimensional features comprised shape and first-order histogram metrics, while high-dimensional features included texture characteristics derived from various matrices: gray-level co-occurrence matrix (GLCM), gray-level run-length matrix (GLRLM), gray-level size-zone matrix (GLSZM), neighborhood gray-tone difference matrix (NGTDM), and gray-level dependence matrix (GLDM). Additionally, texture features were analyzed in the Gaussian Laplacian filter domain (core sizes ranging from 2.0 to 5.0 mm) and the wavelet filter domain. Section title: 2.4 Feature selection Educational score: 4.12300968170166 Domain: biomedical Document type: Study Language: en To assess the interobserver repeatability of radiomic features, the intraclass correlation coefficient (ICC) was calculated using data from 100 randomly selected patients at center A. Features with ICC values greater than 0.8 were deemed highly consistent and were included for further analysis. To harmonize radiomic features across different MRI scanners, the ComBat method was applied . To address biases introduced by imbalanced data distributions, which could affect model performance, the Synthetic Minority Over-sampling Technique (SMOTE) was applied to increase the number of samples in underrepresented classes, thereby improving model robustness . Subsequently, feature selection was conducted on the harmonized T2WI and CE-T1WI features. Initially, a t -test was employed to filter features with significant correlations to treatment outcomes, retaining those with strong associations. Pearson’s correlation coefficient was then computed to analyze the relationships among these features, retaining features with coefficients above 0.8. Finally, the Least Absolute Shrinkage and Selection Operator (LASSO) regression was applied for further refinement, aiming to reduce dimensionality and select the most relevant features for analysis. Section title: 2.5 Construction of stacking ensemble learning model Educational score: 4.104036808013916 Domain: biomedical Document type: Study Language: en Stacking ensemble learning is an advanced machine learning technique that combines the outputs of multiple base learners using a meta-learner to improve predictive performance. In this study, four traditional machine learning algorithms were employed as base learners: Support Vector Machine (SVM), Random Forest (RF), Multilayer Perceptron (MLP), and Light Gradient Boosting Machine (LightGBM). These base learners formed the first layer of the ensemble model. A Logistic Regression (LR) algorithm was selected as the meta-learner for the second layer due to its strong generalization capabilities and effectiveness in combining the predictions of the base learners, while addressing their biases and reducing overfitting. Hyperparameters for each base learner were optimized using Bayesian optimization, which efficiently searches the hyperparameter space to enhance model performance . The predictive performance of the ensemble model was evaluated by calculating the area under the receiver operating characteristic curve (AUC). In addition, metrics such as accuracy, sensitivity, and specificity were assessed using the maximum Youden’s index to provide a comprehensive evaluation of model performance. The radiomics analysis pipeline is shown in Figure 4 . Section title: 2.6 Statistical analysis Educational score: 3.9285390377044678 Domain: biomedical Document type: Study Language: en All statistical analysis was performed using SPSS (version 26.0) and Python (version 3.10). The kappa test was used to analyze the consistency of patient grouping results between two radiologists (Kappa values in the range of 0.80–1.00, good consistency; 0.40 to 0.79, fair consistency; less than 0.40, poor consistency). Continuous variables were described as mean ± standard deviation and compared by a Mann–Whitney U test or t -test. Categorical variables were summarized as frequencies and percentages using the chi-square test or Fisher’s exact test. The AUC of different models were statistically compared using the DeLong test. A P-value <0.05 was considered statistically significant. Section title: 3.1 Patient characteristics and outcome Educational score: 4.098628997802734 Domain: biomedical Document type: Study Language: en In the study, a total of 354 patients were initially enrolled at Center A. Out of these, 54 patients were excluded due to either incomplete or artifact-ridden imaging data. The remaining cohort comprised 172 patients with a favorable prognosis and 128 patients with an unfavorable prognosis. At Center B, 65 patients were screened, with 5 exclusions for the same reasons. This left 35 patients with a favorable prognosis and 25 with an unfavorable prognosis. The inter-rater reliability between the two radiologists was evaluated using the Kappa statistic, which yielded a value of 0.894 (P < 0.001), reflecting a strong level of agreement. The clinical characteristics of the patients are detailed in Table 2 . Section title: 3.2 Feature selection Educational score: 4.094024181365967 Domain: biomedical Document type: Study Language: en Initially, 2,394 features were initially extracted from the ROIs in T2WI and CE-T1WI. After applying an ICC threshold of 0.8, 2,376 features were retained for further analysis. To address class imbalance, the SMOTE algorithm was employed to increase the number of unfavorable prognosis cases in Center A by 44 instances. The t -test was performed to identify features significantly associated with HIFU prognosis, resulting in 491 features. Then, Pearson correlation analysis was used to filter out features with non-significant correlations, reducing the list to 275 features. Finally, LASSO regression was applied for further feature selection and dimensionality reduction, narrowing the list to 36 features from T2WI and CE-T1WI, which were then used to construct the stacking ensemble learning model. The results of feature selection can be found in Supplementary Material S1 . Section title: 3.3 Performance assessment of different models Educational score: 4.125114440917969 Domain: biomedical Document type: Study Language: en The selected features were utilized to construct models with four base learners. Among these, the MLP model showed superior performance, achieving an AUC of 0.858 (95% CI: 0.756–0.959) on the internal test set and 0.828 (95% CI: 0.726–0.930) on the external validation set. This was followed by SVM, LightGBM, and RF, with internal validation set AUCs of 0.841 (95% CI: 0.737–0.946), 0.823 (95% CI: 0.711–0.934), and 0.750 (95% CI: 0.619–0.881), respectively . Detailed performance evaluations of the models are provided in Table 3 . The integration of logistic regression with these four base learners to form an ensemble model led to a substantial enhancement in AUC, reaching 0.897 (95% CI: 0.818–0.977) on the internal test set and 0.854 (95% CI: 0.761–0.952) on the external validation set . Section title: 4 Discussion Educational score: 4.103505611419678 Domain: biomedical Document type: Study Language: en In this study, a novel multimodal MRI stacking ensemble learning model was developed and independently validated, utilizing DL based automated segmentation and integrating radiomics data from T2WI and CE-T1WI sequences. A diverse array of machine learning algorithms, including SVM, RF, LightGBM, and MLP, served as base learners, while LR was utilized as the meta-learner to construct the ensemble model. This approach notably enhanced the precision of predicting HIFU ablation efficacy for uterine fibroids. Compared to single-algorithm models, the ensemble model exhibited a marked improvement in performance, with AUC values rising to 0.897 (95% CI: 0.818–0.977) in the internal validation cohort and 0.854 (95% CI: 0.759–0.948) in the external validation cohort, thereby underscoring the model’s superior capability in managing complex radiomics data. Section title: 4 Discussion Educational score: 4.0977678298950195 Domain: biomedical Document type: Study Language: en In the field of radiomics, precise image segmentation is crucial as it enhances the accuracy of data analysis and provides a solid foundation for subsequent predictive modeling. Current studies predicting the effectiveness of HIFU therapy often rely on manual contouring . This process is not only time-consuming but also prone to human error. Therefore, incorporating automated segmentation technologies is crucial to enhancing efficiency and minimizing human-related biases. In this study, an automated segmentation model based on V-Net was developed specifically for rapid segmentation of uterine fibroids on T2WI and CE-T1WI. This automated tool substantially reduces the need for human intervention, freeing radiomics analysis from the labor-intensive manual contouring and minimizing biases introduced by human factors. This innovation advances the prognostication of HIFU outcomes towards a more efficient and automated future. Section title: 4 Discussion Educational score: 4.326387882232666 Domain: biomedical Document type: Study Language: en Our study has made notable advancements in the field of multimodal MRI analysis by integrating T2WI and CE-T1WI. This integrated approach shows improved predictive performance with an AUC value of 0.858, compared to AUC values of 0.822 for T2WI and 0.848 for CE-T1WI when used independently . The multimodal integration strategy effectively leverages the complementary information from different MRI sequences, offering a more comprehensive description of fibroid tissue characteristics and thus enhancing the accuracy of predicting HIFU treatment efficacy. T2WI, with its superior contrast resolution, reveals cellular dense areas and fibrotic regions within fibroids. These regions manifests as high signal intensity on T2WI and may affect ultrasound transmission efficiency, while fibrotic tissue might interfere with ultrasound propagation characteristics, impacting the effectiveness of HIFU treatment . Additionally, T2WI can identify degenerative features such as necrosis, ischemia, edema, and calcification within fibroids, all of which significantly influence treatment outcomes . Moreover, CE-T1WI assesses the vascular density and blood flow within fibroids, offering insights into ultrasound energy distribution and the thermal effects of HIFU . Regions with abundant vasculature may lead to the dissipation of ultrasound energy, thereby reducing treatment efficacy . Nevertheless, traditional MRI image analysis faces challenges related to subjective interpretation and difficulties in detecting subtle signal intensity variations. This study addresses these limitations by introducing radiomics methods, which overcome these issues by extracting a large number of quantitative features from MRI scans, thereby offering an objective and precise description of fibroid structural heterogeneity . These features not only enhance the analysis of fibroid morphology but also improve predictive capabilities for HIFU treatment outcomes. Furthermore, the combined use of multimodal MRI radiomic features demonstrates the complementarity among different MRI modalities, which is crucial for revealing the biological characteristics of fibroids. Section title: 4 Discussion Educational score: 4.174151420593262 Domain: biomedical Document type: Study Language: en In the medical field, where precise treatment is essential, the efficient and comprehensive utilization of multimodal MRI data is critical for enhancing predictive model performance. However, conventional single machine learning algorithms often fail to fully harness the potential of these invaluable multimodal radiomics datasets due to their disparate methodologies in feature handling and focal points. To address this challenge, this study employs four machine learning algorithms, each with a distinctive modeling philosophy: SVM, RF, MLP, and LightGBM, as base learners. SVM excels in managing small sample sizes and nonlinear challenges by identifying optimal hyperplanes within complex feature spaces ; RF enhances model stability and mitigates overfitting through ensemble decision trees and a voting mechanism ; LightGBM demonstrates exceptional performance in large-scale data processing by iteratively refining weak classifiers ; and MLP leverages deep neural networks to capture intricate nonlinear features, thereby augmenting feature learning capacity . The study integrated these base models using a meta-learner, LR, to construct a stacked ensemble learning framework. This sophisticated approach not only amalgamates the strengths of diverse models but also rectifies the limitations inherent in individual algorithms with regard to multimodal MRI radiomics features utilization. Consequently, it more effectively utilizes multimodal MRI radiomic features to improve both the accuracy and stability of preoperative predictions for HIFU treatment. This robust and advanced tool improves the precision of preoperative assessments, supports personalized treatment strategies, and is expected to enhance treatment decision-making and patient management in clinical practice. Section title: 4.1 Limitations Educational score: 3.4359240531921387 Domain: biomedical Document type: Study Language: en There are some limitations in this study. First, the sample size is relatively small. Future studies should include a larger cohort of patients to enhance the validity of the findings. Second, the effect of HIFU for uterine fibroids is affected by multiple factors, and future studies should integrate more clinical indicators to further improve the performance and credibility of the model. Section title: 5 Conclusion Educational score: 4.063263416290283 Domain: biomedical Document type: Study Language: en This study developed a radiomics stacking ensemble model based on multimodal MRI, incorporating automatic segmentation techniques to predict the efficacy of HIFU ablation for uterine fibroids. It offers a comprehensive method for quantifying uterine fibroid heterogeneity and serves as a more precise supplementary tool for clinical practice. | Review | biomedical | en | 0.999996 |
PMC11695319 | Section title: Introduction Educational score: 4.1886444091796875 Domain: biomedical Document type: Review Language: en Immunological tolerance is a state of unresponsiveness or an anti-inflammatory response that promotes immune homeostasis and prevents detrimental immune reactions directed toward self-antigens and tissues ( 1 ). Tolerogenic vaccines aim to induce antigen-specific tolerance in conditions where tolerance has failed, or where there is an aberrant inflammatory response toward antigens not associated with danger. Tolerogenic vaccines differ from general immunosuppression or immunomodulation in that they aim to induce specific immune tolerance to the disease-relevant antigens, thereby suppressing the autoimmune response without affecting the immune system as a whole ( 2 , 3 ). Autoimmune disease, transplantation rejection, anti-drug antibody responses, and hypersensitivity all represent conditions where tolerogenic vaccines are promising new therapeutic regimens ( 2 , 3 ). Section title: Introduction Educational score: 4.222146987915039 Domain: biomedical Document type: Review Language: en Tolerogenic vaccines can induce antigen-specific tolerance via effects on key players in peripheral tolerance. Mechanisms of immune tolerance are reviewed extensively elsewhere ( 1 , 4 , 5 ). In brief, master regulators of peripheral tolerance toward specific antigens are regulatory T cells (Tregs) and tolerogenic dendritic cells (tolDCs) ( 1 , 3 – 6 ). Tregs exert their immunoregulatory effects via anti-inflammatory cytokines, direct suppression of conventional T cell (Tcon) proliferation, and by modulating DC maturation and function. TolDCs act by promoting Treg development, suppressing effector T cell responses, and by inducing antigen-specific T cell anergy. Many other cell types can contribute to immunological tolerance, of note are B cells producing anti-inflammatory cytokines ( 7 ) and type 1 regulatory T cells (Tr1 cells) ( 8 ). Section title: Introduction Educational score: 4.0363569259643555 Domain: biomedical Document type: Review Language: en Adjuvants are molecules used to enhance the effect of pharmacological treatments ( 9 ), therefore tolerogenic adjuvants aim to increase anti-inflammatory responses and enhance vaccine efficacy. A schematic overview of the main mode(s) of action for tolerogenic adjuvants described in this review are depicted in Figure 1 . Tolerogenic vaccines use adjuvants in different ways depending on vaccine type. Antigen and adjuvants can be administered either separately or co-delivered, where the co-delivery can range from simultaneous administration of free antigen and adjuvant to intricate delivery systems, fusion molecules, or DNA vectors . Tolerogenic adjuvants can also be used to differentiate tolDCs for cell transfer of antigen-loaded tolDCs . Section title: Introduction Educational score: 4.054749488830566 Domain: biomedical Document type: Review Language: en The tolerogenic adjuvants described in this review are grouped in five categories based on their properties and mechanism of action: general immunosuppressive agents ( Table 1 ), cytokines and neuropeptides ( Table 2 ), vitamins and vitamin derivatives ( Table 2 ), modulators of contact-dependent immune cell signaling ( Table 3 ), and other ( Table 4 ). In tolerogenic vaccines using a combination of adjuvants, tolerance can be mediated by multiple mechanisms ( Table 5 ). Tolerogenic vaccines in human clinical trials are discussed for each category and summarized in Table 6 . Section title: Introduction Educational score: 3.9706485271453857 Domain: biomedical Document type: Other Language: en General immunosuppressive agents include molecules with broad immunosuppressive effects on multiple aspects of the immune system, often by suppressing intracellular signaling pathways to reduce inflammation-induced gene expression . General immunosuppressive tolerogenic adjuvants are used both to promote tolerogenic features on antigen presenting cells (APCs) and to suppress effector T and B cell function. Section title: Introduction Educational score: 4.1266770362854 Domain: biomedical Document type: Review Language: en Cytokines orchestrate the balance between immune activation and regulation, making them valuable tools as tolerogenic adjuvants to shape antigen-specific immune responses. Classical anti-inflammatory cytokines like transforming growth factor β (TGF-β), and interleukin (IL)-10 exert potent immunosuppressive functions affecting many aspects of the immune system, including the capacity to induce tolDCs and Tregs . In addition to classical anti-inflammatory cytokines, cytokines affecting specific immune cell subsets or skewing immune balance can also be harnessed as tolerogenic adjuvants. Section title: Introduction Educational score: 3.509734869003296 Domain: biomedical Document type: Other Language: en Many vitamins are important for maintaining a healthy immune system. Vitamins A and D signal in a hormone-like manner via nuclear receptors and are potent immunoregulators ( 10 ). Some of their immunomodulatory effects are inhibition of T effector cell activation and promotion of tolerogenic APCs . Section title: Introduction Educational score: 4.053564071655273 Domain: biomedical Document type: Study Language: en Modulating the immune synapse, the dynamic interface between APCs and T cells during antigen recognition, offers a promising approach to inducing immune tolerance . By manipulating the immune synapse, one can modify T cell receptor (TCR) signaling strength to skew T cell responses or induce anergy or apoptosis ( 11 ). In addition to TCR signaling, modulation of APC co-stimulatory or co-inhibitory molecules in the immunological synapse can also be utilized to modify the outcome of APC-T cell interactions ( 12 ). . Section title: Introduction Educational score: 2.822641134262085 Domain: biomedical Document type: Other Language: en In addition to the adjuvant types described above, there are several other types of adjuvants. Described here are apoptotic remnant mimics , Toll-like receptor (TLR) agonists and glycans and glycan-binding proteins which may promote tolerogenic immune responses. Section title: Introduction Educational score: 4.033331871032715 Domain: biomedical Document type: Review Language: en Depot adjuvants and delivery systems including cell targeting moieties and nanoparticles/microparticles inherently act as adjuvants since they enhance the immune responses to incorporated antigens ( 2 , 3 , 13 , 14 ). Cell targeting strategies which promote antigen delivery to dendritic cells often have tolerogenic properties ( 3 , 14 ) and can be considered tolerogenic adjuvants. However, in this review we specifically explore adjuvants with tolerogenic properties that actively modify the immune milieu to facilitate shifts in immune cell subsets and/or phenotypes. Section title: Dexamethasone Educational score: 4.403566837310791 Domain: biomedical Document type: Study Language: en Glucocorticoids are potent anti-inflammatory and immunosuppressive agents used for treatment of autoimmune and inflammatory conditions ( 15 – 17 ). They mediate their effects through engagement with the nuclear glucocorticoid receptor inducing transcriptional regulation or rapid non-genomic effects ( 15 , 17 ). In immune cells, the synthetic glucocorticoid dexamethasone suppresses production of most cytokines while increasing production of IL-10, inhibits lymphocyte activation and promotes lymphocyte apoptosis ( 15 ). Glucocorticoids dampen overall T cell activation by interfering with TCR signaling, and evidence suggests that glucocorticoids preferentially suppresses Th1 and Th17 T cells. Glucocorticoid treatment is also associated with increased circulating Tregs ( 15 ). In APCs, glucocorticoids induce tolerogenic features including attenuated DC maturation and reduced expression of MHC class II and co-stimulatory molecules ( 15 , 18 ). Section title: Dexamethasone Educational score: 4.110039710998535 Domain: biomedical Document type: Study Language: en Tolerogenic vaccines consisting of antigen delivered with dexamethasone have been successful in murine models of experimental autoimmune encephalitis (EAE) ( 19 – 22 ), atherosclerosis ( 23 ), antigen-induced arthritis (AIA) ( 24 ) and type 1 diabetes (T1D) ( 25 ). Other tolerogenic vaccines use dexamethasone to induce tolDCs, which when loaded with disease relevant antigens and injected reduced disease in models of arthritis ( 26 , 27 ), dust mite allergy ( 28 ) and T1D ( 29 ). The disease inhibition was often accompanied by an increase in Tregs over effector T cells and increase in anti-inflammatory cytokines. However, tolDC-mediated disease suppression in one arthritis model was independent of antigen ( 27 ) and antigen-loaded dexamethasone-derived tolDCs exacerbated a model of T1D while unloaded tolDCs suppressed disease ( 30 ) ( Table 1 ). Section title: Dexamethasone Educational score: 3.884716749191284 Domain: biomedical Document type: Study Language: en Dexamethasone is often used together with other adjuvants to induce tolDCs. TolDC vaccines of dexamethasone in combination with one or more other adjuvants suppressed disease in models of EAE ( 31 , 32 ) and systemic lupus erythematosus (SLE) ( 33 ) ( Table 5 ). Section title: Dexamethasone Educational score: 4.106480598449707 Domain: biomedical Document type: Study Language: en Dexamethasone-containing tolerogenic vaccines have been investigated for human use. A phase I clinical trial investigated transfer of antigen-loaded dexamethasone-treated monocyte-derived DCs for treatment of multiple sclerosis and neuromyelitis optica spectrum disorders, by intravenous injection of tolDC every two weeks for a total of three doses. The tolDC vaccine was well tolerated and there was an increase of regulatory Tr1 cells at 12 weeks of follow-up ( 34 ). In another phase I trial, antigen-loaded autologous monocyte-derived DCs treated with a combination of dexamethasone, the vitamin D derivative calcitriol, and the TLR4 agonist MPLA were administered into inflamed knee joints. The treatment was well tolerated but did not result in reduction in disease severity or consistent immunoregulatory features ( 35 ) ( Table 6 ). Section title: Rapamycin Educational score: 4.096451282501221 Domain: biomedical Document type: Study Language: en Rapamycin is a small molecule inhibitor of mTOR, a kinase that regulates cell growth and metabolism. Immunosuppressive effects of rapamycin includes suppression of the activation and proliferation of conventional T cells, promotion of T cell anergy or deletion, and enhancement of the development and function of Tregs ( 36 ). Additionally, rapamycin can inhibit the differentiation and maturation of DCs and promote tolDC features ( 37 ). Rapamycin is clinically used as an immunosuppressant for prevention of organ transplant rejection, as well as an anti-cancer drug. Section title: Rapamycin Educational score: 4.146580219268799 Domain: biomedical Document type: Study Language: en Tolerogenic nanoparticle vaccines with antigen and rapamycin have successfully reduced disease in multiple models of autoimmunity including arthritis ( 38 ), Alzheimer’s disease ( 39 ), vitiligo ( 40 ), allergic airway disease ( 41 , 42 ), T1D ( 43 ), and EAE ( 44 , 45 ). Rapamycin-containing nanoparticle vaccines also prevented anti-drug antibody responses toward coagulation factor VIII (FVIII) ( 45 , 46 ), uricase ( 47 ), adalimumab (anti-TNFα) ( 47 ), and adenoviral vectors used in gene therapy ( 48 – 50 ). Administration of rapamycin without nanoparticle carrier suppressed OVA-induced skin graft rejection ( 51 ) and anti-FVIII anti-drug antibodies in mice ( 52 ). Observed immunological alterations in studies of tolerogenic vaccines with rapamycin included increased Tregs, decreased levels of inflammatory cytokines, and/or decreased co-stimulatory molecules on DCs ( Table 1 ). Section title: Rapamycin Educational score: 4.079087734222412 Domain: biomedical Document type: Study Language: en Two phase Ia and phase Ib clinical trials were conducted investigating administration of rapamycin together with pegadricase, a biological drug used for the treatment of gout, finding that rapamycin suppressed the development of anti-drug antibodies in a dose dependent manner ( 53 ). Their following phase III trial demonstrated high response rate, safety, and clinically meaningful reduction in serum urate ( 54 ). Another phase I clinical trial administered rapamycin-containing nanoparticles together with AAV8 capsid used in adenoviral gene therapies, leading to reduced development of anti-AAV8 antibodies compared to subjects receiving capsid without rapamycin (press release) ( Table 6 ). Section title: Calcineurin inhibitors Educational score: 4.198665142059326 Domain: biomedical Document type: Study Language: en Cyclosporine A and FK506 (tacrolimus) are calcineurin inhibitors which suppress downstream nuclear factor of activated T cell (NFAT) signaling and IL-2 production ( 55 ). Tolerogenic vaccination with cyclosporine A and autoantigens prevented the development of T1D in pre-diabetic mice and promoted Tregs and tolDCs ( 56 ). Likewise, tolerogenic vaccination with FK506 co-delivered with DNA encoding autoantigen suppressed EAE ( 57 ), and adoptive transfer of antigen-loaded FK506-induced tolDCs reduced disease in a model of CIA ( 58 ), in both studies with a reduction in Th17 responses ( Table 1 ). Section title: NFκB inhibitors Educational score: 4.000032901763916 Domain: biomedical Document type: Study Language: en Nuclear factor kappa B (NFκB) is a transcription factor inducing expression of pro-inflammatory genes including cytokines, cell adhesion molecules, and immunoreceptors. Inhibition of NFκB suppresses T cell responses and induces immunoregulatory features in APCs ( 59 ). Section title: NFκB inhibitors Educational score: 4.118605613708496 Domain: biomedical Document type: Study Language: en Tolerogenic vaccination with antigen-loaded tolDCs treated with NFκB inhibitor BAY 11-7082 suppressed disease in AIA ( 60 ), and antigen-loaded tolDCs treated with NFκB inhibitor andrographolide reduced anti-drug antibodies in hemophilia A mice ( 61 ). A nanoparticle vaccine loaded with antigen and A20, an anti-inflammatory protein inhibiting NF-κB activation ( 62 ), suppressed Th2 responses and reduced disease in an asthma model ( 63 ) ( Table 1 ). Section title: NFκB inhibitors Educational score: 4.048908233642578 Domain: biomedical Document type: Other Language: en A phase I clinical trial investigated antigen-loaded autologous Bay11-7082-treated tolDCs in rheumatoid arthritis. The vaccine reduced effector T cells and increased Tregs, together with a reduction in inflammatory cytokines. The treatment did not induce disease flares and lead to decreased rheumatoid arthritis DAS28 score ( 64 ) ( Table 6 ). Section title: Kynurenine and AhR agonists Educational score: 4.193378448486328 Domain: biomedical Document type: Study Language: en Kynurenine is an immunoregulatory tryptophan metabolite signaling via the aryl hydrocarbon receptor (AhR). AhR signaling can influence T cell differentiation and function of APCs to favor expansion of Tregs ( 65 , 66 ). Tolerogenic vaccination with nanoparticles loaded with autoantigen and the AhR agonist ITE (2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester), suppressed disease in models of EAE ( 67 , 68 ) and T1D ( 69 ), and tolerogenic vaccination with kynurenine and antigen-expressing phages prevented hyperglycemia in a model of T1D ( 70 ) ( Table 1 ). Section title: Other immunosuppressive agents Educational score: 4.125978469848633 Domain: biomedical Document type: Study Language: en Janus kinase (JAK) and signal transducer and activator of transcription proteins (STAT) signaling induces immune cell activation and cytokine production ( 71 ). Tolerogenic vaccination with autoantigen-loaded tolDCs treated with JAK/STAT inhibitors tofacitinib and BD750 suppressed disease, reduced Th1 and Th17 responses, and increased Tregs in models of EAE ( 72 , 73 ) ( Table 1 ). Section title: Other immunosuppressive agents Educational score: 4.102142810821533 Domain: biomedical Document type: Study Language: en Rosiglitazone is an anti-diabetic drug that activates peroxisome proliferator–activated receptor gamma (PPARγ). In immune cells, rosiglitazone can inhibit inflammatory cytokines and promote tolerogenic APCs ( 74 ). TolDC vaccination with antigen-loaded, rosiglitazone-treated tolDCs suppressed disease in CIA ( 75 ) ( Table 1 ), and autoantigen-loaded tolDCs treated with rosiglitazone in combination with dexamethasone and cobalt (III) protoporphyrin suppressed a murine model of SLE ( 33 ) ( Table 5 ). Section title: Other immunosuppressive agents Educational score: 4.076009750366211 Domain: biomedical Document type: Study Language: en Inhibition of the protein kinase glycogen synthase kinase 3 (GSK-3) in immune cells leads to reduced inflammatory cytokine production and increased IL-10 ( 76 ). Vaccination with antigen-loaded tolDCs treated with GSK-3β inhibitor K313 suppressed disease in EAE ( 77 ) ( Table 1 ). Section title: Other immunosuppressive agents Educational score: 4.138024806976318 Domain: biomedical Document type: Study Language: en Prostaglandin I2 (PGI2) is a lipid signaling mediator most known for its vasodilating and anti-thrombotic effects. PGI2 also has anti-inflammatory properties and protective effects in allergy and asthma ( 78 , 79 ). Tolerogenic vaccination with antigen-loaded tolDCs treated with PGI2 analog iloprost reduced disease in ovalbumin-induced asthma ( 80 ). Additionally, direct delivery of iloprost and antigen using a hydrogel suppressed antigen-induced lung inflammation and increased the frequency of antigen-specific Tregs ( 80 ) ( Table 1 ). Section title: TGF-β Educational score: 4.282962799072266 Domain: biomedical Document type: Study Language: en TGF-β is a strongly immunosuppressive cytokine, because genetic deficiency of TGF-β leads to fatal autoimmunity ( 81 ). TGF-β is an immunosuppressive cytokine with multiple effects on the immune system: it promotes Treg development and function, inhibits B and T cell proliferation, suppresses differentiation of Th1 and Th2 cells, and induces tolDCs ( 82 ). However, when combined with specific other cytokines, TGF-β may trigger T cells to differentiate into non-regulatory phenotypes such as Th17 effectors in presence of IL-6 and Th9 in presence of IL-4 ( 82 ). Section title: TGF-β Educational score: 4.134209632873535 Domain: biomedical Document type: Study Language: en A tolerogenic nanoparticle vaccine containing autoantigen and TGF-β reduced disease and immune cell activation in EAE ( 83 ). TolDC vaccination with autoantigen-loaded tolDCs cultured in presence of TGF-β or TGF-β receptor agonist suppressed disease in a CIA model ( 84 ) and reduced anti-drug antibodies toward FVIII ( 85 ), together with increases in Tregs and IL-10 ( Table 2 ). Section title: TGF-β Educational score: 4.069332122802734 Domain: biomedical Document type: Study Language: en In combination with other adjuvants, tolerogenic vaccines using both TGF-β and IL-10 suppressed disease in CIA ( 86 ) and reduced anti-drug antibodies toward FVIII ( 87 ). Tolerogenic vaccination with microparticles containing GM-CSF and TGF-β1 alongside nanoparticles with antigen and vitamin D suppressed EAE and T1D ( 88 – 92 ) and microparticles loaded with TGF-β, retinoic acid, and autoantigens suppressed T1D ( 93 ) ( Table 5 ). Section title: TGF-β Educational score: 4.0809006690979 Domain: biomedical Document type: Study Language: en A tolerogenic DNA vaccination with autoantigen-encoding plasmids in combination with plasmids for either TGF-β, IL-10, and or IL-2 suppressed disease in EAE ( 94 ) and T1D ( 95 ) ( Table 5 ). A phase I clinical trial is registered for this tolerogenic DNA vaccine to evaluate vaccine safety in patients with T1D ( Table 6 ). Section title: IL-10 Educational score: 4.232239723205566 Domain: biomedical Document type: Study Language: en IL-10 is a powerful immunosuppressive and anti-inflammatory cytokine, absence of which causes spontaneous colitis in mice ( 96 ). IL-10 suppresses antigen presentation and inflammatory cytokine production by APCs and simultaneously increases their release of anti-inflammatory mediators. In CD4 + T cells, IL-10 inhibits proliferation and cytokine production and promotes the development of regulatory Tr1 cells ( 97 ). Section title: IL-10 Educational score: 4.056185722351074 Domain: biomedical Document type: Study Language: en Tolerogenic vaccination with antigen-loaded tolDCs engineered to express IL-10 suppressed disease in mouse models of T1D and asthma ( 98 , 99 ), and antigen-loaded tolDCs cultured in presence of IL-10 reduced disease in EAE ( 100 ). Tolerogenic vaccination with nanoparticles containing IL-10 and antigen suppressed disease in EAE ( 101 ), and DNA vaccines encoding IL-10 and antigen suppressed disease in EAE and T1D models ( 102 , 103 ) ( Table 2 ). Section title: IL-10 Educational score: 4.121502876281738 Domain: biomedical Document type: Study Language: en A probiotic vaccine of Lactococcus lactis (L. lactis) genetically engineered to secrete IL-10 and pro-insulin administered together with anti-CD3 ameliorated disease and increase Tregs in models of T1D ( 104 – 106 ) ( Table 5 ). The L. lactis probiotic vaccine has been studied in human clinical trials where results from phase Ib and IIa studies demonstrated treatment to be safe, metabolic variables were either stabilized or improved, and antigen-specific CD8 + T cells were reduced ( 107 ) ( Table 6 ). Section title: IL-2 Educational score: 4.078696250915527 Domain: biomedical Document type: Study Language: en IL-2 mediates T cell survival, differentiation, and proliferation. IL-2 is specifically required for Treg homeostasis and suppression of autoimmunity and genetic deletion results in systemic autoimmunity in mice ( 108 ). In addition, recent studies showed that low-dose IL-2 treatment induces the expansion of Treg cells and had efficacy in numerous mouse models and some early efficacy in clinical trials of T1D, graft-vs-host disease and SLE. Different types of tolerogenic vaccination with IL-2 treatment in combination with antigen exposure suppressed disease in models of EAE ( 109 ), experimental autoimmune uveitis (EAU) ( 110 , 111 ), T1D ( 112 ), delayed-type hypersensitivity (DTH) ( 112 ), and reduced development of anti-drug antibodies toward FVIII in hemophilia A ( 113 ). Overall, the vaccines led to increased Tregs and anti-inflammatory cytokines ( Table 2 ). Section title: IL-2 Educational score: 4.059267044067383 Domain: biomedical Document type: Study Language: en Tolerogenic vaccines using IL-2 in combination with rapamycin expanded Tregs and suppressed disease in models of T1D ( 114 , 115 ), EAE ( 116 ), and primary biliary cholangitis ( 114 ). Furthermore IL-2 in combination with Retinoic Acid suppressed EAE and EAU ( 117 ). These combination vaccines expanded Tregs or induced antigen-specific Tr1 cells ( Table 5 ). Section title: IFN-β Educational score: 4.164449691772461 Domain: biomedical Document type: Study Language: en Interferon beta (IFN-β) is a type I interferon with immunomodulatory properties, used therapeutically for multiple sclerosis (MS). IFN-β reduces T cell activation, promotes Tregs and induces tolDCs ( 118 , 119 ). Tolerogenic vaccines comprised of autoantigen and IFN-β suppressed murine and rat models of EAE via the induction of neuroantigen-specific, suppressive CD25 + Tregs ( 120 , 121 ) ( Table 2 ). In addition, mice treated with IFN-β while receiving autoantigen-loaded vitamin D-treated tolDCs, further suppressed disease in a model of EAE ( 122 ) ( Table 5 ). Section title: GM-CSF Educational score: 4.0019402503967285 Domain: biomedical Document type: Study Language: en In addition to being a growth factor and chemokine, GM-CSF possesses anti-inflammatory effects. Administration of GM-CSF leads to a reduction in disease severity in several animal models of autoimmune disease ( 123 ), and GM-CSF promotes development and function of both tolDCs and Tregs ( 123 , 124 ). Section title: GM-CSF Educational score: 4.133502006530762 Domain: biomedical Document type: Study Language: en Tolerogenic vaccines with antigen-GM-CSF conjugates using neuropeptide autoantigens have been used to treat EAE in mice and rats, accompanied by increased Tregs ( 125 – 129 ). Additionally, GM-CSF is used for differentiation of DCs for most tolDC vaccines. Although most tolDC vaccines use additional adjuvants, also without other adjuvants transfer of antigen-loaded GM-CSF differentiated tolDCs suppressed murine EAU ( 130 ) ( Table 2 ). A hydrogel/microparticle vaccine incorporating GM-CSF and TLR9 agonist CpG with autoantigen suppressed disease in a model of T1D ( 131 ) ( Table 5 ). Section title: Other cytokines and chemokines Educational score: 4.102670192718506 Domain: biomedical Document type: Study Language: en IL-35 is a potent inducer of Tregs and regulatory B cells, and it can inhibit the proliferation and function of effector Th1 and Th17 cells. IL-35 has been shown to be protective against autoimmune disease and IL-35 treatment have been able to suppress disease in multiple models of autoimmunity and chronic inflammation ( 132 ). In tolerogenic vaccines, tolDCs engineered to overexpress IL-35 and loaded with disease relevant antigen suppressed EAE and DTH ( 133 , 134 ) ( Table 2 ). Section title: Other cytokines and chemokines Educational score: 4.164064884185791 Domain: biomedical Document type: Study Language: en IL-27 is an immunoregulatory cytokine which can promote tolerance by supporting development of Tregs and Tr1, antagonizing development of Th2 and Th17 cells, and by increasing co-inhibitory receptor expression on APCs ( 135 ). Tolerogenic vaccination by adoptive transfer of IL-27-conditioned antigen-loaded DCs led to a significant amelioration of disease and reduction in Th1 and Th17 cells in murine EAE ( 136 ) ( Table 2 ). Section title: Other cytokines and chemokines Educational score: 4.0283660888671875 Domain: biomedical Document type: Study Language: en IL-4 promotes type 2 immunity and suppresses Th1 polarization. Treatment with IL-4 suppressed disease severity in models of EAE and arthritis ( 137 , 138 ). Tolerogenic DNA vaccines encoding IL-4 and antigen suppressed murine models of CIA, EAE, and T1D ( 139 – 142 ) ( Table 2 ). Section title: Other cytokines and chemokines Educational score: 4.1202192306518555 Domain: biomedical Document type: Study Language: en Hepatocyte growth factor (HGF) is a cytokine with pleiotropic effects, including the promotion of tolDCs ( 143 ). In a model of EAE, systemic HGF ameliorated disease and tolerogenic vaccination with HGF-treated antigen-loaded DCs mediated functional recovery in mice with established EAE and suppressed T cell mediated inflammation ( 144 ) ( Table 2 ). Section title: Other cytokines and chemokines Educational score: 4.143764495849609 Domain: biomedical Document type: Study Language: en Vasoactive intestinal peptide (VIP) is a peptide functioning as a neurotransmitter in the central and peripheral nervous systems and has multiple effects, including immune modulation. VIP reduces the release of inflammatory cytokines, stimulates production of IL-10 and TGF-β, and decreases the co-stimulatory activity of APCs ( 145 ). Tolerogenic vaccination with antigen-loaded VIP-treated tolDCs led to amelioration of CIA and EAE, accompanied by increased levels of Tr1 cells ( 146 ) ( Table 2 ). Section title: Other cytokines and chemokines Educational score: 4.157129287719727 Domain: biomedical Document type: Study Language: en TNF-related apoptosis-inducing ligand (TRAIL) is a cytokine that induces apoptosis and activation of NFκB. TRAIL has immunoregulatory effects demonstrated by the exacerbated development of autoimmunity in TRAIL-deficient mice ( 147 ). Tolerogenic vaccination with DCs engineered to co-express TRAIL and antigen reduced antigen-specific T cell responses and disease symptoms in models of EAE ( 148 , 149 ) ( Table 2 ). Section title: Cytokine silencing Educational score: 4.10301399230957 Domain: biomedical Document type: Study Language: en Just as the addition of anti-inflammatory or immunoregulatory cytokines can promote tolerogenic responses, silencing of inflammatory cytokines can also be effective. Silencing of B cell activating factor (BAFF), an essential cytokine for both T and B cell activation ( 150 ), in TolDCs using siRNA suppressed murine CIA and promoted Tregs ( 151 ) ( Table 2 ). Section title: Vitamin D Educational score: 4.152439594268799 Domain: biomedical Document type: Study Language: en Vitamin D is primarily known for its role in calcium homeostasis and bone health, but it is also immunoregulatory. Having low levels of vitamin D is associated with increased susceptibility to a variety of infectious and autoimmune diseases. Vitamin D suppresses T cell activation, skews T cell differentiation away from Th17 while promoting Tregs and Th2 responses, and promotes tolerogenic DC features, including low surface expression of co-stimulatory molecules and decreased production of inflammatory cytokines ( 10 , 152 ). Vitamin D signals via a nuclear receptor and exert immunoregulatory effects by regulating gene expression ( 10 ). Section title: Vitamin D Educational score: 4.110340118408203 Domain: biomedical Document type: Study Language: en Many tolerogenic vaccines using vitamin D are tolDC vaccines. Vitamin D-treated tolDCs loaded with antigen or antigen-encoding mRNA reduced disease and promoted immunoregulatory cells and cytokines in murine models of EAE ( 153 – 156 ). The vitamin D-treated tolDCs had reduced expression of MHC class II, co-stimulatory molecules, and pro-inflammatory cytokines, and induced less T cell proliferation compared to DCs that were untreated ( 153 , 154 ) ( Table 2 ). TolDC vaccines using a combination of vitamin D and dexamethasone suppressed murine models of arthritis ( 26 ) and T1D ( 29 ) ( Table 5 ). Section title: Vitamin D Educational score: 3.9861338138580322 Domain: biomedical Document type: Study Language: en Tolerogenic nanoparticle vaccines with vitamin D have been examined in T1D ( 157 , 158 ), where the nanoparticles reduced disease incidence and increased Tregs or tolDCs in vivo . Separate delivery studies of vitamin D and antigen have been studied in EAE ( 159 , 160 ) and DTH ( 161 ), in all studies reducing disease severity and inflammation ( Table 2 ). Section title: Vitamin D Educational score: 4.108878135681152 Domain: biomedical Document type: Study Language: en Vitamin D-containing tolerogenic vaccines have been investigated in human clinical trials. In a phase IIa trial of latent autoimmune diabetes in adult patients were treated with daily oral Vitamin D and monthly injections of antigen-alum. The trial demonstrated safety and stable β-cell function and metabolic control at 5 months follow-up ( 162 ). Two phase I clinical trials have been registered to test tolerogenic vaccination with antigen-loaded vitamin D-treated monocyte-derived DCs in multiple sclerosis ( 163 ). Likewise, a phase I clinical trial tested monocyte-derived DC loaded with proinsulin and treated with Vitamin D and dexamethasone in T1D. The study showed the treatment was safe and lead to reduce proinsulin specific CD8+ T cells and increased ICOS + CCR4 + TIGIT + Tregs ( 164 ). Another phase I trial investigated liposomes containing collagen II and calcitriol, the active form of vitamin D, for the treatment of rheumatoid arthritis. The calcitriol-antigen-liposomes led to reduced pathogenic T cells and expansion of antigen-specific PD1 + T cells ( 165 ) ( Table 6 ). Section title: Retinoic acid Educational score: 4.201015472412109 Domain: biomedical Document type: Study Language: en Retinoic acid is an immunoregulatory vitamin A metabolite, which like vitamin D signals via a nuclear receptor ( 10 ). Retinoic acid promotes the development and function of Tregs while inhibiting the differentiation and activation of effector Th1 and Th17 cells. DCs and macrophages can produce retinoic acid to support Treg induction and maintenance, and the retinoic acid-producing capacity of DCs is further enhanced upon retinoic acid exposure ( 10 , 166 ). Section title: Retinoic acid Educational score: 4.133817195892334 Domain: biomedical Document type: Study Language: en Tolerogenic vaccination with liposomes incorporating retinoic acid and autoantigen converted pathogenic autoantigen specific Th17 cells to Tr1 cells and suppressed disease in EAE ( 167 ). Tolerogenic vaccination with retinoic acid, IL-2, and autoantigen suppressed EAE and EAU and pathogenic Th17 and Th1 responses ( 117 ). Prophylactic tolerogenic vaccination with retinoic acid, TGF-β and autoantigen inhibited the incidence of T1D in mice ( 93 ), and in another study treatment of mice with autoantigen and retinoic acid in combination with IL-4 suppressed EAE ( 168 ) ( Tables 2 , 6 ). Section title: T cell modulation Educational score: 4.182868957519531 Domain: biomedical Document type: Study Language: en CD3 is the invariant chain of the TCR. Anti-CD3 monoclonal antibodies suppresses disease in numerous animal models of autoimmunity, and anti-CD3 is an FDA approved treatment to delay early onset type 1 diabetes. The exact mechanism of anti-CD3-mediated immune suppression is unclear but proposed mechanisms include prevention of T cells from recognizing their antigens and the induction of anergy or apoptosis in activated T cells while sparing Tregs ( 169 ). Tolerogenic vaccination with anti-CD3 treatment in combination with an antigen-expressing lentiviral vector suppressed T1D and induced autoantigen-specific Tregs ( 170 ). Anti-CD3 is also a component of the previously described probiotic vaccine ( 104 – 106 ) ( Tables 3 , 5 ). Section title: T cell modulation Educational score: 4.183112144470215 Domain: biomedical Document type: Study Language: en CD4 is a glycoprotein on helper T cells which primarily functions as a TCR co-receptor. Non-depleting anti-CD4 therapy has been shown to suppress autoimmunity and graft rejection by modulating the function of CD4 + T cells by blocking T cell activation and promoting Treg differentiation and suppressor functions ( 171 ). A tolerogenic vaccine comprised of aluminum hydroxide (alum), FVIII, and non-depleting anti-CD4 prevented development of anti-drug antibodies in mice ( 172 ). Another tolerogenic vaccine using treatment with depleting anti-CD4 antibodies followed by antigen administration suppressed disease in a murine model of EAU via the induction of antigen-specific Tregs ( 173 ) ( Table 3 ). Section title: T cell modulation Educational score: 4.130310535430908 Domain: biomedical Document type: Study Language: en “Tregitopes” are peptides derived from IgG that are recognized by a subset of natural Tregs. When presented in MHCII, these peptides activate Tregitope-specific Tregs and suppression of effector T cell responses to co-delivered antigens. Administration of nanoparticles with antigen and tregitopes decreased incidence and severity of T1D in mice ( 174 ). Likewise, co-administration of autontigen with Tregitope-albumin fusion proteins decreased incidence and reverse mild T1D ( 175 ) ( Table 3 ). Section title: T cell modulation Educational score: 4.165193557739258 Domain: biomedical Document type: Study Language: en Invariant natural killer T cells (iNKT cells) are immunoregulatory T cells important for preventing autoimmune reactions. The glycolipid α-galactosylceramide (α-GalCer) is a strong inducer of iNKT cells and has been shown to suppress disease in multiple animal models of autoimmunity ( 176 ). A tolerogenic vaccine comprised of lipid nanoparticles carrying autoantigen and α-GalCer prevented the development of diabetes in prediabetic mice ( 177 ) ( Table 3 ). Section title: Modulation of the immunological synapse Educational score: 4.105387210845947 Domain: biomedical Document type: Study Language: en Including MHC class II-targeting molecules in a tolerogenic vaccine ensures delivery to APCs and may disrupt the immunological synapse. A tolerogenic vaccine with antigen conjugated to antibody fragments (nanobodies) targeting MHC class II suppressed disease in EAE. When combined with dexamethasone, the vaccine also overcame the inflammation associated with antigen exposure ( 178 ) ( Table 5 ). Section title: Modulation of the immunological synapse Educational score: 4.148727893829346 Domain: biomedical Document type: Study Language: en Co-stimulatory signals, such as CD80 and CD86, are necessary for T cell activation by APCs. T cell recognition of antigen on MHC II without co-stimulation results in anergy or apoptosis. Abatacept, a cytotoxic T-lymphocyte associated protein 4 (CTLA-4)-Fc fusion protein blocks CD80 and CD86 and is FDA approved for the treatment of autoimmune arthritis ( 179 ). Tolerogenic vaccination with nanoparticles displaying abatacept and carrying autoantigen and dexamethasone suppressed EAE ( 180 ) ( Table 5 ). Section title: Modulation of the immunological synapse Educational score: 4.1392059326171875 Domain: biomedical Document type: Study Language: en Signaling via CD40-CD40L induces activation and pro-inflammatory cytokine production in both B cells, T cells, and APCs, and CD40L-blockade reduced disease in numerous animal models of autoimmunity ( 181 ). Tolerogenic vaccination with FVIII and anti-CD40 prevented subsequent development of anti-FVIII antibodies during rechallenge ( 182 ) ( Table 3 ). Section title: Modulation of the immunological synapse Educational score: 4.172018051147461 Domain: biomedical Document type: Study Language: en Other approaches include genetic modification of co-stimulatory signals. Antigen delivered with a CRISPR-Cas9 plasmid and guide RNAs toward CD80, CD86, and CD40 disrupted co-stimulation by DCs, reduced inflammatory cytokines, increased Tregs, and suppressed disease in a model of T1D ( 183 ) ( Table 5 ). Administration of a DNA vector encoding membrane-bound autoantigen together with a B7.1/CD40L mutant fusion protein binding to CTLA-4 but not CD28, providing co-inhibitory but not co-stimulatory signals, reduced disease incidence in murine model of T1D ( 184 ) ( Table 3 ). Section title: Modulation of the immunological synapse Educational score: 4.165256500244141 Domain: biomedical Document type: Study Language: en Intercellular adhesion molecule 1 (ICAM-1) is a cell surface glycoprotein most known for its role in leukocyte migration. Inhibition of ICAM-1 can block T cell activation and induce tolerance by disrupting T cell-APC interactions, inhibiting co-stimulation, promoting PD-L1 expression, and by inducing T cell anergy or exhaustion ( 185 ). A tolerogenic vaccine comprised of hyaluronic acid with autoantigen and an ICAM-1 inhibitory peptide suppressed disease in EAE ( 186 – 188 ). In another tolerogenic vaccine, fusion molecules of antigen and ICAM-1 inhibitory peptide prevented the development of T1D ( 189 ) and suppressed EAE ( 190 ) ( Table 3 ). Section title: Modulation of the immunological synapse Educational score: 4.121855735778809 Domain: biomedical Document type: Study Language: en OX40 is a TNF receptor superfamily member expressed by activated T cells and resting Tregs, which acts as a co-stimulatory molecule promoting cell proliferation ( 191 ). Tolerogenic vaccination of prediabetic mice with antigen and an OX40 agonistic antibody reduced diabetes incidence and increased antigen specific Tregs ( 192 ) ( Table 3 ). Section title: Activation of inhibitory receptors Educational score: 4.121880531311035 Domain: biomedical Document type: Study Language: en Programmed cell death protein 1 (PD-1) is an immune checkpoint molecule that induces T cell apoptosis and suppresses conventional T cell activation and favors development of Tregs ( 193 ). Treatment with tolDCs engineered to express PD-L1 and MOG reduced antigen-specific T cell responses and suppressed MOG induced EAE but not MBP induced EAE ( 148 , 149 ) ( Table 3 ). Section title: Activation of inhibitory receptors Educational score: 4.126893520355225 Domain: biomedical Document type: Study Language: en B and T lymphocyte attenuator (BTLA) is an inhibitory receptor structurally related to PD-1, activation of which leads to the suppression of T cell activation ( 194 ), and BTLA-expressing DCs promoted Treg development ( 195 ). Adoptive transfer of bone marrow derived DCs treated with a nanoparticle containing antigen and a BTLA-encoding plasmid suppressed EAE ( 196 ) ( Table 3 ). Section title: Activation of inhibitory receptors Educational score: 4.159967422485352 Domain: biomedical Document type: Study Language: en Fas is a death receptor inducing apoptosis upon binding to Fas ligand (FasL). A tolerogenic vaccine using FasL-conjugated microparticles containing monocyte chemotactic protein-1 (MCP-1) recruited T cells and then induced their apoptosis. When coupled with respective antigens, the microparticles could suppress EAE and prevented development of T1D in pre-diabetic mice ( 197 ). ( Table 5 ) A tolerogenic vaccine aiming for engagement of multiple inhibitory receptors for immune suppression used microparticles displaying surface PD-L1-Fc, anti-Fas, and self-marker CD47, and containing TGF-β. The vaccine led to reduced T cell infiltration and EAE suppression ( 198 , 199 ) ( Table 5 ). Section title: Activation of inhibitory receptors Educational score: 4.153730392456055 Domain: biomedical Document type: Study Language: en CD22 and siglec G are inhibitory receptors inhibiting B cell receptor (BCR) signaling, thus suppressing B cell responses ( 200 ). Tolerogenic vaccination with liposomes displaying antigen and CD22/siglec G ligands induced antigen-specific tolerance in mice and reduce development of anti-drug antibodies toward FVIII in a model of hemophilia A ( 201 ) and reduced antigen-specific antibody production ( 202 ) ( Table 3 ). Encapsulation of rapamycin in CD22L autoantigen liposomes suppressed arthritis in mice ( 203 – 205 ) ( Table 5 ). Section title: Modulators of apoptotic pathway signaling Educational score: 3.9369540214538574 Domain: biomedical Document type: Study Language: en Apoptotic cells are cleared by phagocytic cells via anti-inflammatory mechanisms, which in part is mediated by phosphatidylserine exposed on the apoptotic cell surface ( 206 ). Therefore, phosphatidylserine liposomes, O-phospho-L-serine (OPLS), or pro-apoptotic factors have been used as tolerogenic adjuvants to reduce antigen immunogenicity. Section title: Modulators of apoptotic pathway signaling Educational score: 4.187808990478516 Domain: biomedical Document type: Study Language: en In hemophilia A, both co-delivery of FVIII with OPLS and tolerogenic nanoparticle vaccination with FVIII encapsulated in phosphatidylserine liposomes led to a reduction in anti-drug antibodies toward FVIII in mice ( 207 , 208 ), and antigen-containing phosphatidylserine liposomes reduced disease incidence in a model of T1D ( 209 ). In addition, phosphatidylserine liposomes loaded with collagen peptide and the immunomodulator leflunomide, which inhibits the mitochondrial enzyme dihydroorotate dehydrogenase preventing uridine synthesis, suppressed CIA in mice ( 210 ). Furthermore, a DNA vaccine encoding antigen and the pro-apoptotic protein BAX, promoting apoptosis in cells expressing antigen, suppressed T1D via modulation of APC function and promotion of Treg development ( 211 , 212 ) ( Table 4 ). Section title: TLR agonists Educational score: 4.026141166687012 Domain: biomedical Document type: Study Language: en TLR agonists are well known inflammatory stimuli and often used as adjuvants in immunogenic vaccines to enhance the immune response to the target antigen ( 213 ). However, signaling through microbial pattern recognizing receptors might also protect from development of autoimmunity. Dose and administration of the TLR agonist affects if the response is immunogenic or tolerogenic, it is believed that a short-term, high-dose stimulation will result in immunogenic responses whereas low-dose, repeated stimulation results in tolerance ( 214 ). Section title: TLR agonists Educational score: 4.1402482986450195 Domain: biomedical Document type: Study Language: en TLR4 agonist LPS is often used in cultures of DCs to induce maturation and enhance their antigen presenting capacity. Antigen-loaded LPS-treated DCs suppressed EAE while non-treated DCs did not ( 215 ). A recombinant fusion protein of autoantigen and flagellin A, TLR5 agonist, induced production of IL-6 and IL-10 in DCs and reduced T cell-driven inflammation in a murine model of intestinal allergy ( 216 , 217 ). Co-delivery of flagellin B and antigen reduced disease in models of allergy ( 218 , 219 ) and a fusion protein of antigen and flagellin B reduced disease and IgE responses in allergy ( 220 ) ( Table 4 ). Section title: TLR agonists Educational score: 4.123254776000977 Domain: biomedical Document type: Study Language: en TLR9 agonist CpG DNA has been used in different tolerogenic vaccines in combination with other adjuvants. Treatment with a hydrogel vaccine containing CpG DNA, antigen, and GM-CSF prevented and delayed disease onset in pre-diabetic mice, and the inclusion of CpG DNA enhanced efficacy compared to GM-CSF alone ( 131 ). Tolerogenic vaccination with antigen, CpG DNA, and heat shock protein 60 induced an antigen-specific increase in IL-10 production and reduced disease severity in a model of arthritis ( 221 ) ( Table 5 ). Section title: Glycans and glycan-binding proteins Educational score: 4.000657081604004 Domain: biomedical Document type: Study Language: en The use of glycans in tolerogenic vaccine design can mediate tolerance by targeting antigen to APCs expressing receptors for these glycans, promoting its uptake and processing, while concurrently inducing immunoregulatory effects in DCs ( 14 , 222 , 223 ). Section title: Glycans and glycan-binding proteins Educational score: 4.211918354034424 Domain: biomedical Document type: Study Language: en β-glucan is a polysaccharide naturally occurring in cell walls of plants, bacteria, and fungi, and binds to Dectin-1 on myeloid cells. In a model of T1D, treatment with β-glucan and antigen led to increased protection from disease compared to treatment with β-glucan or antigen alone, and promoted tolDC features and increased Tregs ( 224 ). Conjugates of allergens to mannan, targeted these antigens to APCs expressing mannose and C-type lectin receptors, and promoted a tolerogenic response in comparison to native allergens in vitro and in vivo ( 222 , 223 ). Skin-prick tests with mannan-conjugated grass pollen allergoids caused less inflammation than native allergens in patients with grass pollen allergy ( 225 ), and immunization of mice with mannan-allergoid conjugates led to tolerogenic responses and increase in Foxp3 + Tregs compared to native antigen ( 225 , 226 ) ( Table 4 ). Mannan-allergoid conjugates have been tested for dust mite and grass pollen allergens in two phase II clinical trials, showing improvement in nasal provocation test ( 227 , 228 ) ( Table 6 ). Section title: Glycans and glycan-binding proteins Educational score: 4.117554664611816 Domain: biomedical Document type: Study Language: en Galectin-1 is a glycan-binding protein with diverse functions, including modulation of DCs and T cell responses ( 229 ). In a study of EAE, treatment of DCs with galectin-1 led to tolDC differentiation that suppressed EAE when loaded with relevant autoantigen ( 230 ). The disease suppression was dependent on IL-27 and IL-10 induced by galectin-1 ( 230 ) ( Table 4 ). Section title: Discussion Educational score: 4.237026214599609 Domain: biomedical Document type: Review Language: en Tolerogenic vaccines are promising experimental treatments for a wide range of conditions, including autoimmune disease, anti-drug antibody responses, transplantation rejection, and hypersensitivity ( 3 , 6 ). Successful reintroduction of immune tolerance via tolerogenic vaccination would have numerous benefits over traditional immunosuppression or immune modulation. First, tolerance could be durable as tolerogenic vaccines may deplete or inactivate pathogenic cells, while concurrently inducing long lived suppressive Tregs and/or regulatory B cells which can self-renew and persist ( 231 , 232 ). Second, tolerogenic vaccines engaging antigen-specific Treg responses may engender bystander suppression and infectious tolerance to suppress autoimmune responses to unknown antigens ( 3 , 233 ). Third, tolerogenic vaccines may have efficacy with minimized toxicity as they modulate the antigen-specific response, leaving the rest of the immune system intact. Together these characteristics could constitute a functional cure. Section title: Discussion Educational score: 4.134842395782471 Domain: biomedical Document type: Review Language: en Although tolerogenic adjuvants are not always necessary in tolerogenic vaccines ( 3 , 234 , 235 ) addition of tolerogenic adjuvants have the potential to greatly enhance the efficacy of tolerogenic vaccines by several mechanisms. Immunosuppressive or anti-inflammatory tolerogenic adjuvants promote an anti-inflammatory environment upon antigen encounter, thereby reducing the risk of unwanted inflammatory responses, anaphylaxis or disease exacerbation when re-introducing disease-relevant antigens. Immunomodulatory tolerogenic adjuvants can steer the antigen-specific immune response in desired direction, and cell-targeting adjuvants ensure vaccine delivery to intended cell types and minimizes off-target effects. Additionally, tolDC transfer is a common tolerogenic vaccine modality, but this type of tolerogenic vaccine is associated with high costs and difficulties of standardization across patients. Therefore, tolerogenic vaccines using adjuvants to deliver antigen to and modulate DCs in vivo may represent a more feasible treatment option. Section title: Discussion Educational score: 4.115062713623047 Domain: biomedical Document type: Study Language: en A major limitation to the development of tolerogenic vaccines is a lack of understanding of the autoantigen pools that drive autoimmune diseases. Few autoimmune diseases have limited and well-defined antigen pools, while most autoimmune disease have numerous, poorly defined or undefined antigen pool, and disease antigens may change through time or differ across patients. Therefore, initial clinical trials using tolerogenic vaccines have focused on conditions which have relatively defined antigen pools such as celiac disease, pemphigus vulgaris, T1D and anti-drug antibody responses. However, preclinical data suggest that induction of tissue-specific Tregs may circumvent the need-to-know exact antigens involved in disease as these vaccine-induced tissue-specific Tregs can traffic to the inflamed tissue and exert suppressive functions via bystander suppression or infectious tolerance to suppress immune responses to unknown antigens involved autoimmune disease ( 3 , 233 ). Utilizing adjuvants to expand or enhance Treg responses, may therefore enable further application of tolerogenic vaccines also in autoimmune diseases with complex autoantigen pools. Section title: Discussion Educational score: 4.180022716522217 Domain: biomedical Document type: Study Language: en Another unknown is if tolerogenic vaccines that induce tolDCs and/or Tregs can suppress preexisting pathogenic B cell that were licensed by CD4 T cell and have limited ongoing interactions with either Tregs or TolDCs. Therefore, tolerogenic vaccines might have to address B cells separately. This could be achieved by B cell targeting or B cell suppression, such as by adjuvants signaling via Siglec G and CD22 or using adjuvants with effects on both B and T cell responses. This approach could be combined with a tolerogenic vaccine design acting on tolDC and/or T cells to prevent further activation of novel pathogenic B cell clones. Section title: Discussion Educational score: 3.710791826248169 Domain: biomedical Document type: Other Language: en In conclusion, tolerogenic vaccines may be the therapeutics of the future for autoimmune and inflammatory conditions. Tolerogenic adjuvants are powerful tools with capacity to both enhance antigen-specific tolerance as well as reduce the risk of unwanted inflammatory responses or off-target effects. | Review | biomedical | en | 0.999996 |
PMC11695320 | Section title: Introduction Educational score: 4.155217170715332 Domain: biomedical Document type: Study Language: en Infectious diseases cause major costs and constraints to livestock production. It is estimated that over 600 million people globally depend on livestock, and represent up to 70% of the population in the most marginal areas ( 1 , 2 ). In low and middle income countries (LMICs), particularly in sub-Saharan Africa (SSA), cattle are infected by a range of economically important and endemic diseases, each with distinct epidemiological properties ( 3 ). Among those affecting cattle in SSA, tick-borne pathogens collectively represent some of the most impactful including multiple piroplasma species such as Theileria and Babesia , and Rickettsia such as Anaplasma and Ehrlichia . These organisms are termed ‘haemopathogens’ as they are predominantly bloodstream based in the mammalian host ( 4–7 ). Haemopathogens are a particularly important group of pathogens in Africa and contribute to high levels of mortality in cattle ( 8 , 9 ). Section title: Introduction Educational score: 4.4831647872924805 Domain: biomedical Document type: Study Language: en Bovine piroplasmosis is caused by the tick-borne Babesia and Theileria spp. which are arguably some of most economically important livestock parasites ( 10 , 11 ). Within these genera, some species are highly pathogenic, such as Theileria parva , the main agent of East Coast fever and corridor disease, which kills over 1 million cattle each year in SSA ( 11–13 ). Other Theileria species circulating in Africa include T. mutans , T. taurotragi , T. sergenti/buffeli/orientalis (referred to as the T. orientalis complex) and T. velifera ( 14 ), all of which are considered to be either less pathogenic or not pathogenic in cattle, and are reported to cause benign, moderate or asymptomatic theileriosis ( 15 ). In Africa, bovine babesiosis is caused by Babesia bovis and B. bigemina ( 16 ). While B. bigemina is more prevalent, B. bovis infection results in a greater disease burden because of the neurological symptoms associated with infection ( 17 ). Anaplasmosis and Ehrlichiosis are also highly important tick-borne ruminant diseases in SSA. Heartwater, caused by Ehrlichia ruminantium ( 18 , 19 ), and infections with Anaplasma centrale , A. phagocytophilum , A. bovis and A. marginale, are widespread ( 20 , 21 ). The latter is known to be pathogenic to domestic ruminants, particularly to high producing dairy cattle ( 22 ). Section title: Introduction Educational score: 4.015689373016357 Domain: biomedical Document type: Study Language: en Historically, understanding of these pathogens has progressed through investigation and study of one pathogen at a time using a range of diagnostic approaches to detect the specific pathogens, including microscopy ( 8 ), antibody-based techniques such as ELISA ( 23 , 24 ), and several PCR methods including conventional PCR, reverse line blot (RLB)-PCR, quantitative qPCR and multiplex PCR ( 25–29 ). However, it is clear that co-infections are both common and important, and that pathogen combinations can work together (synergistically) or against each other (antagonistically) ( 30 , 31 ). Additionally, many pathogen infections cause immunosuppression, such that infection with one pathogen increases the chance that a host will be infected with another. In SSA, co-infection, including with species of the same genus or group of genera, are common in livestock ( 8 , 32 ) and relevant at both the level of small holder farmers and commercial ranching operations. Section title: Introduction Educational score: 4.345332622528076 Domain: biomedical Document type: Study Language: en If we are to understand the complex interactions that occur in the multiple-pathogen disease ecosystem in sub-Saharan livestock—which potentially influence the prevalence of other pathogens, cattle productivity, and the effectiveness of interventions—it is essential to use tools that can identify multiple pathogens. These tools will be crucial for generating the requisite data at scale. Next Generation Sequencing (NGS) technologies represent powerful approaches to enable the investigation of the population genetics, ecology and dynamics of pathogen communities across a range of taxonomic scales ( 33 ). NGS targeting one region of DNA provides millions of sequences with low error rates, making it feasible to investigate species diversity and prevalence in large populations ( 34–36 ). One approach involves PCR amplification using custom-designed oligonucleotide primers targeting the 16S/18S ribosomal DNA of the pathogen, a suitable target due to the highly conserved sequences flanking this region. The significant species-specific variation within this region then allows for the discrimination between species ( 37 , 38 ), and potentially also facilitates the distinction between strains or subtypes (depending on coverage) ( 36 , 39 ). This approach has previously been applied to Theileria and Babesia ( 32 , 36 , 40–45 ), demonstrating the robustness of the NGS method. The amplified PCR products can then be subjected to multiplex amplicon sequencing that enables high throughput data generation from hundreds or thousands of samples, with a custom-designed bioinformatics pipeline facilitating downstream deconvolution of data per sample and allocation of sequenced reads to pathogen species. Using multiplexed barcoded primer combinations, up to 384 samples can be processed simultaneously on a single Illumina MiSeq flow cell, significantly reducing costs. Section title: Introduction Educational score: 4.134708404541016 Domain: biomedical Document type: Study Language: en The aim of this study was to develop and validate a novel high-throughput diagnostic tool capable of detecting a range of important haemopathogens in livestock, which would remove the need to individually screen for one pathogen at a time. Compared to previous studies, we have now also included pathogens from the Ehrlichia and Anaplasma genera. We hereafter refer to the platform as the Haemabiome tool, and see its potential utility for the in-depth elucidating of co-infections, allowing simultaneous detection of many of the most important vector-borne livestock pathogens (the tool specifically targets any species in the Theileria , Babesia , Erhlichia and Anaplasma genera) with high confidence. This sequencing approach will also allow the detection of both pathogenic and low/non-pathogenic subspecies within the genera, and opens up the study of co-infections in ways previously not possible ( 32 , 36 ). It also has future application potential to clinical diagnostics in the field. Section title: Positive control samples Educational score: 4.050868034362793 Domain: biomedical Document type: Study Language: en DNA from laboratory cultured strains of pathogens or verified single-pathogen infections were used as single-species control samples to evaluate the specificity of primer pairs, and this panel included Anaplasma phagocytophilum , Ehrlichia ruminantium , Babesia bigemina , Babesia bovis , Theileria annulata , Theileria mutans , Theileria parva and Theileria taurotragi (for more details see Table 1 ). Section title: Field samples Educational score: 4.114816665649414 Domain: biomedical Document type: Study Language: en Samples originally collected from the Infectious Diseases of East African Livestock (IDEAL) calf cohort study conducted in Kenya from 2007 to 2009 were used ( 8 ). In this cohort study, calves were followed from birth up to 1 year old. These calves did not receive any preventive vaccines or treatments during this period in order to measure true cumulative pathogen exposure. For the purposes of tool validation, we examined samples from a subset of 31 calves from the IDEAL study in three categories: (1) nine animals with clinical episodes that survived, (2) nine animals without clinical episodes that survived and (3) 13 animals not categorised. In addition, a subset of eight of these IDEAL samples, underwent sequencing across different runs to assess reproducibility of results. These particular samples will be henceforth referred to as “repeated samples.” A total of 279 Kenyan cattle blood samples from the above-mentioned animals were tested in this study ( Supplementary Table S1 ). These subsets were selected as they can provide insight into tool validation with respect to calves that are more likely to be infected (‘clinical disease’ cohort), as well as the degree of infection/co-infection occurring in calves that did not undergo clinical episodes (‘no clinical episodes’), and animals that were previously diagnosed to be infected with African trypanosomes, to assess diversity in animals infected with a pathogen known to cause immunosuppression ( 46 ) and therefore potentially predispose to multiplicity of infection (‘trypanosomes’). Section title: DNA extraction Educational score: 4.057647705078125 Domain: biomedical Document type: Study Language: en DNA was extracted from 100 μL of blood originally collected from the calves in the IDEAL study and stored in the ILRI liquid nitrogen biobank, using the Qiagen DNA blood and tissue kit according to the manufacturer’s recommendation, with the modification of incubating blood with lysis buffer for 30 min to allow full lysis of the cattle blood. A control extraction was also carried out using distilled water as a substrate, and this was subsequently used as a negative control to test for any cross-contamination that may have occurred during DNA extraction. Section title: Primer design and validation Educational score: 4.088532447814941 Domain: biomedical Document type: Study Language: en For the amplification of parasites from the Theileria and Babesia genera, the V4 hypervariable fragment (378–424 bp) of the 18S rRNA gene was targeted. Primers were adapted from the primers previously described by Bishop et al. ( 47 ); RLB-F ( 28 ) and RLB-R2 ( 48 ) ( Table 2 ). Section title: Primer design and validation Educational score: 4.094763278961182 Domain: biomedical Document type: Study Language: en For Anaplasma sp. and Ehrlichia sp., primers were designed in the homologous regions of the variable regions of 16S rDNA based on the 23 Anaplasma sp. and Ehrlichia sp. sequences obtained from NCBI database ( Supplementary Table S2 ) and their specificity was confirmed by blasting the primer sequence against the NCBI database. Different combinations of primers, predicted to give an amplicon size of between 100 and 600 bp, were tested using a panel of control samples with known pathogen content, to validate their ability to amplify samples with Anaplasma sp. and Ehrlichia sp., but not other haemopathogens. Based on the validation results, the AE-F4 and AE-R3 primer pair was selected ( Table 2 ). Section title: Generation of 16S/18S rDNA PCR amplicons for sequencing Educational score: 4.100180149078369 Domain: biomedical Document type: Study Language: en In order to generate amplicons with unique identifiers ready for sequencing, two rounds of PCR were performed. The first-round targeted amplification of the genus specific sequences of the 16S/18S rDNA locus using primers which included adapter sequences to facilitate the second round of PCR. The second round of amplification was performed to anneal the Nextera II multiplex identifier tags (MID) and sequencing primers ( Supplementary Table S3 ). Section title: Generation of 16S/18S rDNA PCR amplicons for sequencing Educational score: 4.126103401184082 Domain: biomedical Document type: Study Language: en The two sets of genus specific PCRs; Theileria/Babesia (referred to hereafter as “ThBa”) and Anaplasma / Ehrlichia (referred to hereafter as “AnEh”) were conducted in separate 20 μL reactions, each containing 4 μL Phusion high-fidelity PCR master mix (New England Biolabs), 0.2 μL HF Phusion Taq Polymerase, 10 mM dNTPs, 0.6 μL DMSO, 10 μM of each forward and reverse primer, and 2 μL of template DNA. Cycling conditions were as follows: Theileria/Babesia : 98°C (30 s), 25 cycles of 98°C (10 s), 60°C (30 s) and 72°C (30 s), with 10 min at 72°C; Anaplasma/Ehrlichia : 98°C (30 s), 25 cycles of 98°C (10 s), 64°C (20 s), 72°C (20 s) with 10 min at 72°C. For initial assessment of amplification, some of the amplified products were subjected to electrophoresis in a 1.5% agarose gel in TAE buffer and visualised under ultraviolet light. PCR products were then purified using AMPure XP magnetic beads (Beckman Coulter) according to manufacturer’s instructions. Section title: Generation of 16S/18S rDNA PCR amplicons for sequencing Educational score: 4.245405197143555 Domain: biomedical Document type: Study Language: en The second round PCR was performed by using combinations of 16 forward (N502 to N511, N513 to N522) and 12 reverse barcoded primers (N701 to N715) ( Supplementary Table S3 ) so that each individual sample in a pool was assigned a unique barcode combination. PCRs were performed using the same reaction mix as described for the first round PCR, except for the addition of 10 μM barcoded forward and reverse primers, and 1 μL of first round PCR product as a template. Cycling conditions were as follows: Theileria/Babesia : 98°C (30 s), 10 cycles of 98°C (10 s), 64°C (30 s), 72°C (30 s), and 10 min at 72°C. Anaplasma/Ehrlichia : 98°C (30 s), 10 cycles of 98°C (10 s), 64°C (20 s), 72°C (20 s), and 5 min at 72°C. 10 μL of each sample was pooled to create a master sequencing library pool. Then, 100 μL from each pool were loaded onto a 1.5% agarose gel. The products were then purified using by Qiagen QIAquick gel extraction kit (Qiagen) and subsequently with AMPure XP magnetic beads (1X), following the manufacturer’s instructions (Beckman Coulter). Each pool was then quantified using Qubit (Invitrogen) according to the manufacturer’s instructions. Finally, 70 μL of purified pool, at a concentration of 50 nM for AnEh and 22 nM for ThBa, was submitted for sequencing on an Illumina MiSeq platform using a 500-cycle paired-end reagent kit (MiSeq Reagent Kits v2, 2 × 250 bp paired-end reads) with 10% PhiX Control v3 . Each of the pools were sequenced in a separate lane. Section title: Generation of 16S/18S rDNA PCR amplicons for sequencing Educational score: 4.026786804199219 Domain: biomedical Document type: Study Language: en The first round PCR product of all samples was assessed for positivity on an agarose gel. Based on the results observed on the gel, we proceeded to create two different libraries for each infection. The first library consisted of samples that showed the expected positive band on the gel, and included selected positive and negative control samples. The second library comprised samples that did not show any visible bands by gel electrophoresis in the first PCR. These samples were pooled separately, also including selected positive and negative samples, and eight repeated samples from the first library to assess the haemobiome tool’s reproducibility. Section title: Species-specific PCR validation Educational score: 4.13956356048584 Domain: biomedical Document type: Study Language: en In order to compare the haemabiome results with data derived from well characterised species-specific assays, specific primers targeting T. parva sporozoite microneme-rhoptry surface antigen (p104) and the A. bovis 16S rRNA gene were amplified by a two-step semi-nested PCR using forward and reverse primers as previously described ( 49 , 50 ). For T. parva p104, the first round PCR was performed using 20 μL reactions containing 4 μL Phusion high-fidelity PCR master mix (New England Biolabs), 0.2 μL HF Phusion Taq Polymerase, 10 mM dNTPs, 10 μM of each forward and reverse primer, 11.8 μL ddH20 and 1 μL of template. p104 first round PCR cycling conditions were 98°C (30 s), [98°C (10 s), 62°C (30 s), 72°C (20 s min)] for 30 cycles, and 2 min at 72°C. For second round PCR, 1 μL of first PCR product as template with the same conditions, except the annealing temperature which was 66°C. Section title: Species-specific PCR validation Educational score: 4.063019275665283 Domain: biomedical Document type: Study Language: en For the A. bovis 16S gene, PCR was performed using 20 μL reactions containing 10 μL Q5 High-Fidelity Reaction Buffer (New England Biolabs), 10 μM of each forward and reverse primer, 7 μL ddH20 and 1 μL of template. The first round PCR cycling conditions were 98°C (1 min), [98°C (1 min), 56°C (1 min), 72°C (1:30 min)] for 35 cycles and 5 min at 72°C. Section title: Species-specific PCR validation Educational score: 3.958750009536743 Domain: biomedical Document type: Study Language: en For the second round PCR, 1 μL of first PCR product was used as template with PCR conditions of 98°C (30 s), [98°C (10 s), 62°C (30 s), 72°C (30 s)] for 30 cycles and 2 min at 72°C. Section title: Bioinformatic analysis Educational score: 4.266300201416016 Domain: biomedical Document type: Study Language: en Amplicon libraries were submitted to Edinburgh Genomics Core facility to generate 250 bp paired end sequencing reads on Illumina MiSeq v2 platform. The raw sequencing data has been submitted to the European Nucleotide Archive and is available under the project accession number PRJEB79313. Sequencing reads were deconvoluted into data from individual samples based on the barcoding combinations. To ensure data quality, the raw sequences were evaluated using Fastqc and poor quality reads with a Phred score below 28 were removed using Sickle ( 51 ). The resulting high quality paired end reads were merged using Flash ( 52 ) to produce extended amplicon sequences. These merged sequences were then separated based on the presence of primers used for AnEh and ThBa. Within data from each sample, sequences that were 100% identical were grouped into clusters. However, clusters were excluded from further analysis if they met the following criteria: (1) they represented a single copy sequence read, (2) they were predicted to be formed as a PCR chimera from two other more frequent cluster sequences within the same sample, or (3) if they differed by only one nucleotide from a cluster sequence present in the same sample with higher read counts and a fold change of 3, as these clusters may have arisen from sequencing or PCR errors and could not confidently be defined as distinct. The remaining clusters were compared to the SILVA database v138.1 (18S/16S rRNA) using BLAST ( 53 ). Following the data from the control samples, specific criteria were established to define positive infections in each sample. These measures comprised the following rules: (a) establishing read frequency thresholds of 1,000 for AnEh and 500 for ThBa mapped clusters , (b) setting percentage identity thresholds of 99 and 97% for AnEh and ThBa, respectively, and (c) ensuring sequence lengths were within the expected ranges of 160–162 bp for Anaplasma / Ehrlichia and 330–378 bp for Theileria / Babesia , respectively . Section title: Sequence alignment, sequences and phylogenetic analyses Educational score: 4.066563606262207 Domain: biomedical Document type: Study Language: en The obtained partial 16S/18S sequences were aligned with reference sequences from Genbank and existing literature listed in Supplementary Tables S2, S4 , using Clustal W with Bioedit software version 7.2.5 ( 54 ). Phylogenetic trees were constructed using trimmed partial 16/18S gene sequences from both the IDEAL sample data and aforementioned reference sequences. Section title: Sequence alignment, sequences and phylogenetic analyses Educational score: 4.126126766204834 Domain: biomedical Document type: Study Language: en To determine the best available evolutionary models for Bayesian inference (BI), we used JModelTest version 0.1.1.40 ( 55 ) and selected models based on Akaike Information Criterion (AIC). The Bayesian analysis was performed using MrBayes 3.2.6_168 ( 56 ) through the phylogeny web service. 1 The Bayesian analysis processed two simultaneous independent runs with four chains each proceeding for 1,000,000 generations. Trees were sampled every 250 generations. The first 25% of iterations were excluded as burn-in. Resulting trees were combined to create a majority-rule consensus tree, from which posterior probabilities were obtained. A Paracoccus sp. and the Hemalivia stellate sequences were used as outgroups for constructing the AnEh and ThBa trees, respectively, following the study by Chiuya et al. ( 57 ). Section title: Statistical analysis Educational score: 4.060154914855957 Domain: biomedical Document type: Study Language: en Species-specific nested PCR results were taken as the “gold standard” and compared with the Miseq results. Using the constructed gold standard, sensitivity and specificity were estimated using the “medcalc” software version 19.2.6 ( 58 ). Agreement between the different diagnostic tests (species-specific nested PCR and Miseq) assessing the presence of A. bovis and T. parva was calculated. A kappa measure of agreement test was performed to compare the performance of the two tests; κ -value < 0 indicates no agreement beyond chance. A κ -value between 0.41 and 0.60 indicates a moderate level of agreement while a κ -value between 0.81 and 0.99 indicates almost perfect agreement ( 59 ). Other statistical analyses were carried out using R version 3.5.1. Section title: Comparison of the Haemabiome tool with known controls, replicates and species-specific primers Educational score: 4.056739330291748 Domain: biomedical Document type: Study Language: en To validate the specificity of the Haemabiome tool, we performed the two PCRs using the primer sets described in Table 2 on the set of negative control samples (water negative controls processed using the DNA extraction protocol). We used these results to set the selected filtering thresholds for each pathogen genus (AnEh <1,000 reads and ThBa <500 reads, respectively). Although there are small numbers of reads present in the negative control samples, these are generally well below thresholds and can be confidently assigned as ‘false positives.’ Section title: Comparison of the Haemabiome tool with known controls, replicates and species-specific primers Educational score: 4.122648239135742 Domain: biomedical Document type: Study Language: en Moreover, for the AnEh primers several bacterial genera were present in some negative controls, which were previously reported as contaminants in DNA extraction kits, PCR and other laboratory reagents. This included Acinetobacter , Actinomyces , Bradyrhizobium , Corynebacterium , Cutibacterium , Delftia , Moraxella , Ralstonia , Stenotrophomonas , Streptococcus , and Xanthomonas ( 60 , 61 ). Although detected with very few reads (<100), other bacterial genera such as Gordonia and Nocardioides could also be considered potential contaminants of the 16S rDNA NGS process for the AnEh primers. However, some negative control samples from the AnEh PCRs did generate read numbers that approached the threshold of 1,000 reads . When checked, the sequences in these reads aligned with “uncultured bacterium,” indicating clear false positives. These data provided confidence that the tool was not generating false positives above the defined threshold . Section title: Comparison of the Haemabiome tool with known controls, replicates and species-specific primers Educational score: 3.9976022243499756 Domain: biomedical Document type: Study Language: en Sequencing replicates were carried out using the species-specific positive control DNA ( Table 1 ) to validate the consistency, accuracy and reliability of deep amplicon sequencing. Read numbers for each replicate set are shown in Figure 2 . The results showed that the Haemabiome tool successfully amplified and detected all positive control samples, albeit with varying read numbers between different sequencing runs . Section title: Comparison of the Haemabiome tool with known controls, replicates and species-specific primers Educational score: 4.070366382598877 Domain: biomedical Document type: Study Language: en To assess reproducibility, eight field samples were subjected to repeat testing in different sequencing runs for both AnEh and ThBa primer sets . In each distinct run, the same pathogen species were consistently detected within each tested sample, and the number of reads linked to each species showed similar relative patterns between repeats. This consistent detection and comparable pattern of read counts indicated that the Haemabiome tool successfully identified the presence of the same pathogen species in both runs in similar proportions, confirming reliability and accuracy of the approach for each pathogen genus across independent sequencing runs. Section title: Comparison of the Haemabiome tool with known controls, replicates and species-specific primers Educational score: 4.05124044418335 Domain: biomedical Document type: Study Language: en Finally, as part of the validation we utilised known species-specific primers for the selected individual pathogens, to confirm the Miseq data generated using the Haemabiome tool and the IDEAL samples. For the amplification of A. bovis a nested PCR targeting the species-specific16S gene was used ( 50 ). For T. parva we used a species-specific nested PCR targeting the T. parva p104 gene ( 49 ). Section title: Comparison of the Haemabiome tool with known controls, replicates and species-specific primers Educational score: 4.112593650817871 Domain: biomedical Document type: Study Language: en A total of 279 samples from the 31 animals were screened. Of these, 179 (64.8%) were positive for A. bovis by species-specific nested PCR, compared to 138 (49.4%) samples testing positive using the Haemabiome tool ( Table 3 ). The calculated κ -value, which measures the agreement between the two tests, for A. bovis detection, indicated a substantial level of agreement ( κ = 0.65) ( Table 4 ). Section title: Comparison of the Haemabiome tool with known controls, replicates and species-specific primers Educational score: 4.104288578033447 Domain: biomedical Document type: Study Language: en A total of 83 (29.7%) samples were positive by the p104 nested PCR for T. parva , while only 23 (8.2%) samples were positive on the Miseq assay ( Table 3 ). The calculated kappa value of 0.26 indicated only slight or weak agreement between the PCR and Miseq tests for the presence of T. parva infections ( Table 4 ). Section title: Comparison of the Haemabiome tool with known controls, replicates and species-specific primers Educational score: 4.111442565917969 Domain: biomedical Document type: Study Language: en Using the species-specific PCR results as the gold standard we estimated the sensitivity and specificity of the Haemabiome tool ( Table 4 ). The sensitivity and specificity of the Haemabiome tool were estimated as 77.9 and 96%, respectively for A. bovis , resulting in an overall accuracy of 82.4%. On the other hand, the Haemabiome tool had an estimated sensitivity of 22.9% and specificity of 98% for T. parva , resulting in an accuracy of 81.7%. Section title: Comparison of the Haemabiome tool with known controls, replicates and species-specific primers Educational score: 3.138491153717041 Domain: biomedical Document type: Study Language: en This suggests that the Haemabiome tool may be significantly less sensitive than the nested PCRs, which is to be expected given the high sensitivity of the latter tests. Section title: Comparison of the Haemabiome tool with known controls, replicates and species-specific primers Educational score: 4.041916847229004 Domain: biomedical Document type: Study Language: en Figure 4 shows a comparison of the Haemabiome tool with species-specific PCR per calf and at different sampling times to confirm if the same sample is positive or negative for both approaches. This figure clearly demonstrates that samples detected as positive by the gold standard method were almost all positive by Miseq . Additionally, the results obtained from the field samples illustrate the power of the Haemabiome tool in accurately identifying the multiplicity of infections. Section title: Gel electrophoresis analysis Educational score: 4.124932765960693 Domain: biomedical Document type: Study Language: en Initially all 279 samples were assessed by gel electrophoresis for presence of visible bands after the first round of PCR. Out of these, 108 displayed the expected band on the gel for AnEh, of which 89 were also positive for on the Miseq sequencing results, while the remaining 19 samples were negative. By comparison, 100 of the 171 AnEh samples that did not exhibit the anticipated band on gel electrophoresis, were positive for AnEh in the Miseq data, despite the lack of visible band in gel electrophoresis. Out of the 279 samples tested using the ThBa PCR 156 were positive based on gel electrophoresis, with 146 of those samples confirmed positive on Miseq sequencing. However, among the 123 samples classified as negative by gel electrophoresis, 73 were identified as positive for ThBa infection using the Haemabiome tool ( Supplementary Table S5 ). Section title: Overall diversity of Anaplasma and Ehrlichia identified by the Haemabiome tool in the IDEAL cohort field samples Educational score: 4.151826858520508 Domain: biomedical Document type: Study Language: en Among the detected Anaplasma species, A. bovis was found in 49.5% (138/279) of samples, and A. platys in 42.6% (119/279), making them the most abundant species in this sample set. Uncultured Anaplasma sp. Saso was the third most detected species (25.1%; 70/279) followed by Candidatus Anaplasma boleense, A. phagocytophilum (1.4%; 4/279) and Uncultured Anaplasma sp. and Anaplasma sp. clone ZJ06 (1%, 3/279). In terms of Ehrlichia , E. minasensis (or E. canis ) was the most abundant species, detected in 6.4%, (18/279) of samples, followed by E. ruminantium at 2.5% (7/279). Overall, 67.7% (189/279) of samples were positive for any Anaplasma / Ehrlichia infection ( Table 5 ). Section title: Overall diversity of Anaplasma and Ehrlichia identified by the Haemabiome tool in the IDEAL cohort field samples Educational score: 4.109092712402344 Domain: biomedical Document type: Study Language: en The sequences from the 138 Miseq positive samples within the Anaplasma genus resolved into 7 distinct clades . The first Anaplasma clade consisted of all A. phagocytophilum , A. phagocytophilum related and Candidatus Anaplasma boleense sequences. This included three sequences from the present study that shared 100% similarity with a previously published Candidatus Anaplasma boleense isolate and four sequences with previously published A. phagocytophilum sequences . Section title: Overall diversity of Anaplasma and Ehrlichia identified by the Haemabiome tool in the IDEAL cohort field samples Educational score: 4.144824028015137 Domain: biomedical Document type: Study Language: en The second clade is represented by 70 sequences obtained from samples that share 100% nucleotide identity with Uncultured Anaplasma sp. Saso strain , including sequences previously isolated from Kenya by Okal et al. ( 62 ). The third clade comprised a number of A. bovis isolates with 138 (i.e., all those positive on the AnEh PCR) sequences from this study, with 100% identity to a published A. bovis from Okal et al. ( 62 ) in Kenya . A small clade with three identical study sequences were also 100% identical to a sequence of Uncultured Anaplasma sp. clone ZJ06 strain isolated from a cow in China. Section title: Overall diversity of Anaplasma and Ehrlichia identified by the Haemabiome tool in the IDEAL cohort field samples Educational score: 4.043804168701172 Domain: biomedical Document type: Study Language: en A fifth clade consisted of 119 study sequences, which had previously been confirmed as A. platys or A. platys-like sequences. This group included isolates from the study conducted by Okal et al. ( 62 ) , as well as a Candidatus Anaplasma camelii sequence derived from a Kenyan camel. Obtained sequences shared 100% nucleotide identity and 99.4% identity with other A. platys sequences , respectively. Section title: Overall diversity of Anaplasma and Ehrlichia identified by the Haemabiome tool in the IDEAL cohort field samples Educational score: 4.156994819641113 Domain: biomedical Document type: Study Language: en A sixth clade was composed of Anaplasma species that were not detected in our field samples, including A. marginale , A. centrale , A. ovis and other uncultured Anaplasma species . A seventh clade comprised previously published E. ruminantium sequences, along with 7 sequences from present study. These sequences grouped into the clade with 100% sequence similarity with the counterparts in GenBank . Finally, the last clade encompassed the E. canis/E. minasensis group, which included 18 study sequences. These sequences demonstrated 100% sequence identity with counterparts in the database from different hosts , and >99% of identity with the other published sequences . Section title: Overall diversity of Theileria and Babesia identified by the Haemabiome tool in the IDEAL cohort field samples Educational score: 4.119537353515625 Domain: biomedical Document type: Study Language: en T. mutans was the most abundant species and detected in 68.1% (190/279) of samples, followed by Theileria sp. strain MSD in 50.9% (142/279) of samples. T. velifera was the third most detected species at 35.5% (99/279). Finally, T. parva and T. taurotragi were detected in 8.2% (23/279) and 7.2% (20/279) of samples, respectively. In contrast to the high detection rate of Theileria species, B. bigemina was found in only 1.1% (3/279) and B. bovis in only 0.3% (1/279) of samples. Overall, 78.5% (219/279) of the samples were identified as hosting one or more Theileria of Babesia species using the Miseq sequencing approach ( Table 5 ). Section title: Overall diversity of Theileria and Babesia identified by the Haemabiome tool in the IDEAL cohort field samples Educational score: 4.133902549743652 Domain: biomedical Document type: Study Language: en The Theileria and Babesia sequences could be grouped into five clades, and the phylogenetic tree is shown in Figure 6 . The first clade contained only an unique sequence from this study, detected in a single sample, which clustered with a B. bovis published sequence with >99.1% nucleotide identity. The second clade included one sequence identified in three samples this study, which had 100% nucleotide similarity with a B. bigemina sequence isolated from zebu cattle in Uganda as reported by Byaruhanga et al. ( 63 ). Section title: Overall diversity of Theileria and Babesia identified by the Haemabiome tool in the IDEAL cohort field samples Educational score: 4.15871524810791 Domain: biomedical Document type: Study Language: en A third clade encompassed one sequence which was detected in 190 study samples, which grouped together with 100% nucleotide identity with previously published T. mutans sequences ; >99.7% identity was observed with other published sequences, and another sequence which was detected in 142 study samples grouped with Theileria sp. strain MSD . A fourth clade contained three distinct sequences, detected in 20 study samples. The first sequence was detected in four samples and showed 99.69% nucleotide identity with published T. taurotragi sequences . The second sequence was detected in 14 samples and showed 100 and 99.73% nucleotide identity with published T. taurotragi sequences . The third sequence was found in two samples and showed 99.38 and 99.07% nucleotide identity with published T. taurotragi sequences . Section title: Overall diversity of Theileria and Babesia identified by the Haemabiome tool in the IDEAL cohort field samples Educational score: 4.107175827026367 Domain: biomedical Document type: Study Language: en Furthermore, a T. parva sequence was detected in 23 study samples, sharing 99.7% nucleotide identity with the published sequences . The final clade contained a T. velifera sequence, detected in 99 study samples, forming a distinct group with 100% identity with counterparts in the database . Theileria species such as T. annulata , T. buffeli, T. orientalis and T. sinensis presented in a separate group, although no sequences from this study aligned with these species . Section title: Discussion Educational score: 4.058281421661377 Domain: biomedical Document type: Study Language: en In the IDEAL project, Bronsvoort et al. ( 8 ) quantified for the first time in an animal population the high diversity of pathogens that a population may have to deal with over time (the first year of life), and the levels of co-infections with key pathogens such as A. marginale , E. ruminantium and T. parva . They also highlighted the need to develop new systems-based approaches to study pathogens in their natural settings, in order to understand the impacts of co-infections on clinical outcomes and to develop new evidence-based interventions that are relevant. The preliminary presentation of the pathogens has hitherto been simply summed across all visits to estimate the proportion of calves with each pathogen (or pathogen/test combination). However, this ignores the dynamics of the order of exposure. Section title: Discussion Educational score: 4.204819679260254 Domain: biomedical Document type: Study Language: en The current reanalysis of a subset of these samples with the novel Haemabiome tool has demonstrated that the diversity of co-infections at any point can be resolved sufficiently to allow more detailed co-infection studies, which require a high-throughput analysis of the pathogen dynamics and distribution. The Haemabiome tool appears to have very high specificity but moderate sensitivity compared to the two nested PCRs used as the gold standard in this dataset. However, this is to be expected given the very high levels of specificity and sensitivity in the nested PCRs used. The nested PCRs use 60 cycles, whereas only 35 cycles were used to generate the sequencing library. Additionally, it has been suggested by previous studies that NGS approaches may not be ideal for absolute quantification. The more limited sensitivity of NGS datasets can be attributed to potential factors such as mixed infections, PCR competition, and PCR suppression caused by the presence of more abundant templates ( 32 ). There is an obvious trade-off in terms of reduced sensitivity with the high throughput nature and ability of the Haemabiome tool to detect the full range of pathogen diversity in the target genera. For example, animals that are carriers for T. parva (a relatively common state of infection, particularly in adult animals) have low levels of parasitaemia, which might fall under the detection level of the Haemobiome tool. Section title: Discussion Educational score: 4.316307544708252 Domain: biomedical Document type: Study Language: en The diversity of tick-borne pathogen communities detected using the Haemabiome tool is well supported by previous studies conducted in western Kenya on cattle from various genus specific PCR ( 57 , 62 , 64 ). For Anaplasma , A. bovis and A. platys were the most abundant species in both our and the previous studies. However, in our study we did not find any A. marginale infections, while the studies noted above detected A. marginale infection, albeit at low prevalence: only 0.6% in Okal et al. ( 62 ) and 4.9% in Chiuya et al. ( 57 ), respectively whereas Peter et al. ( 64 ) detected 31% of A. marginale infections. Okal et al. ( 62 ) did not find any Ehrlichia spp. and Chiuya et al. ( 57 ) only identified E. minasensis . Ehrlichia sequences amplified from samples in our study cluster phylogenetically between E. canis and E. minasensis reference sequences. As E. minasensis is a tick-borne pathogen affecting cattle, cervids, and dogs, and is closely related to the monocytotropic pathogen E. canis ( 65 ), without further investigation it is only possible to classify samples in our study as E. canis / E. minasensis , highlighting the need for a more accurately curated and appropriately diverse reference sequence database in this approach. Further studies are therefore needed to better understand the epidemiology and dynamics of Ehrlichia transmission in livestock in western Kenya. We detected most Theileria species that were previously identified in western Kenya, and T. mutans and T. velifera being the most abundant Theileria species is consistent with previous studies in this region ( 62 , 63 , 66 ). Additionally, we confirmed that the important cattle pathogen T. parva is circulating in this area. We did not find substantial levels of Babesia spp. infection, but overall, our findings are consistent with previous studies confirming theileriosis is the major circulating infectious piroplasm disease of cattle in western Kenya ( 57 , 62 , 66 ). Section title: Discussion Educational score: 3.8723175525665283 Domain: biomedical Document type: Study Language: en Bronsvoort et al. ( 8 ) highlighted that livestock disease and vector control are indispensable for increasing livestock productivity and preventing losses due to diseases, including disease-related morbidity, mortality, and loss of markets for livestock products in Africa. The lack of disease control measures has implications for the effectiveness of strategies aimed at rapidly improving livelihoods dependent on livestock. One route to improving disease control is to increase the accuracy of identification of infected animals, in order to apply targeted treatment. However, this approach is economically viable only if a single assay can detect as many pathogens as possible. Conducting multiple single-pathogen tests is not economically or logistically feasible, particularly in settings where co-infections are the norm. Current diagnostic markers are species or genus specific and difficult (if not impossible) to scale up in a high-throughput manner. Section title: Discussion Educational score: 4.067708969116211 Domain: biomedical Document type: Study Language: en In this study, we have designed a deep amplicon sequencing tool that in principle will enable detection to investigate the species composition of tick-borne pathogens present in cattle. The Haemabiome tool has the power to provide more accurate and reliable quantification of the pathogen community in terms of species diversity, but can also detect previously unanticipated (or untargeted) pathogen species that may play a role in animal disease. Section title: Discussion Educational score: 4.123461723327637 Domain: biomedical Document type: Study Language: en Overall, we describe the development of a high-throughput amplicon sequencing approach targeting tick-borne pathogen genuses of high relevance to cattle health ( Anaplasma , Ehrlichia , Theileria and Babesia species). The tool permitted us to identify the full diversity of tick-borne pathogens circulating, and its reliability and utility was demonstrated on field samples for which data for particular pathogens was already available. The Haemabiome tool will allow us to explore a range of epidemiological questions in the future, through the generation of large-scale data on the diversity of tick-borne pathogens at a population level. Improved understanding of the diversity of pathogen infections encountered, and the potential impact on disease outcome or severity of interactions between such pathogens, will enable a more efficient combating of the multiple infectious disease threats in the African smallholder farm context, with vector-borne diseases amongst the most impactful. | Study | biomedical | en | 0.999996 |
PMC11695322 | Section title: Introduction Educational score: 4.143558025360107 Domain: biomedical Document type: Review Language: en Over 500 million years ago, ancient cold-blooded fishes emerged along with adaptive immunity ( 1 ). Together with immunological memory ( 2 ), they form the basis of natural immunity, and vaccinology. These phenomena manifest as secretion of microbe-specific agglutinins, memory responses to antigen, accelerated rejection of allografts, and acquired protection from reinfection. They are observable and common to all fish including jawless fish ( 3 , 4 ), cartilaginous fish ( 5 – 7 ), and bony fish ( 8 , 9 ), with published reports dating back to over 80 years ago ( 10 ). Nowadays, we routinely observe adaptive immune and memory responses in a variety of fish species ( 11 – 15 ). Studying how these responses manifest promises solutions for preventing disease in fish. However, despite fish immunology coming to maturity, driven by comparative immunology and the importance of certain species to aquaculture, we still face difficulties identifying and studying the cells and mechanisms responsible for these phenomena. Section title: Introduction Educational score: 4.056685447692871 Domain: biomedical Document type: Study Language: en It is especially difficult to identify homologous and functional molecules, cells and mechanisms in teleost fishes due to their rich diversity, evolutionary history and distance. For example, although the single-cell RNA sequencing of grass carp B cells identified plasma cells ( 16 ), there was a glaring absence of any population that corresponds to memory B cells. Fundamentally, it is phenomena such as longevity and antibody secretion ( 11 – 15 , 17 , 18 ) that are observed time and time again rather than any specific phenotypic marker or mechanism. Therefore, it is from this angle that we studied the common carp B cell response, and searched for memory B and plasma cell analogues. Section title: Introduction Educational score: 4.288435935974121 Domain: biomedical Document type: Study Language: en We leveraged our laboratory model of Cyprinus carpio with the myxozoan (Cnidaria) parasite Sphaerospora molnari ( 19 , 20 ). The myxozoans are ancient cnidarians that have co-evolved with and cycle between invertebrate and fish hosts. They cause economically important seasonal outbreaks in both wild and farmed fish stocks. In addition to the historical uncertainty over their phylogeny ( 21 – 23 ), the Myxozoa are also a scientific metazoan oddity due to adaptations such as one species losing the capacity for mitochondrial respiration ( 24 ). Generally, B cells respond strongly to myxozoan infections with proliferation and antibody production ( 25 , 26 ). Myxozoan infections potentially elicit polyreactive antibodies ( 25 ); they induce distinct transcriptional signatures that frequently include il10 upregulation ( 26 – 28 ); infections are ultimately chronic and latent ( 29 ). These features of both the host response and the pathogenesis of myxozoan infections led us to question whether the B cell response elicits antibodies that mitigate disease and produces differentiated memory B and plasma cells that provide long-term protection. In other words, we investigated whether the B cell response is protective and productive. Section title: Introduction Educational score: 4.275160789489746 Domain: biomedical Document type: Study Language: en In the present study, by removing B cells via immunosuppression ( 19 , 30 – 32 ), we provide evidence that B cells and antibodies limit the otherwise severe parasitemia and disease. Although we measured proliferation via incorporation of the thymidine analogue (5-ethynyl-2′-deoxyuridine, EdU) and detected gene signatures that indicate a productive B cell response, there are currently no specific phenotypic markers nor reagents to directly detect memory B cells. Thus, we relied on a single anti-carp IgM monoclonal antibody at our disposal. As described by Shibasaki et al. in a recent report of a germinal center analogue in fish, EdU labels cells engaged in an immune response and germinal center activity ( 33 ). It is through tracing the long-term retention of the thymidine analogue EdU that we directly detected resting memory B cells. A corticosteroid-resistant, enlarged, and head/anterior kidney lymphoid organ-resident B cell population expressing high levels of cytosolic IgM may represent plasma cells that constitutively secrete antibodies after resolution of the acute immune response. In this complex teleost host-myxozoan parasite interaction, humoral memory of a past infection, and protection are achievable. As our methodology is based on fundamental phenotypes of memory B and plasma cells rather than any homologous markers, it is applicable towards the study of these subpopulations and these phenomena in other species. Section title: The B cell compartment expands and produces S. molnari -specific antibodies that limit parasitemia Educational score: 4.1416335105896 Domain: biomedical Document type: Study Language: en To study the role played by B cells and the antibody response in S. molnari infection, we compared B cell-sufficient and B cell-depleted common carp throughout infection with the parasite. We previously revealed the most severe form of S. molnari infection via immunosuppression with the synthetic corticosteroid triamcinolone acetonide ( 19 , 30 – 32 ). Here, we demonstrate that immunosuppression completely depletes the peripheral IgM + B cell population within 3 days of administration and allows us to study the outcome of infection in the absence of B lymphocytes. Section title: The B cell compartment expands and produces S. molnari -specific antibodies that limit parasitemia Educational score: 3.9994962215423584 Domain: biomedical Document type: Study Language: en Fish were either not treated with the corticosteroid (immunosufficient) or immunosuppressed with it (IS). Under each of these two conditions, we further subdivided fish into three groups receiving one of three ten-fold diluted doses of diethylaminoethyl (DEAE) cellulose-purified S. molnari blood-stage parasite cells (BSs) ranging from 2,500,000 to 25,000. Additionally, mock-infected fish were included as a control group. Section title: The B cell compartment expands and produces S. molnari -specific antibodies that limit parasitemia Educational score: 4.169050216674805 Domain: biomedical Document type: Study Language: en Following infection, we measured changes in peripheral blood composition, focusing on expansion of the B cell compartment which is a hallmark of the infection and B cell activation ( 26 ). Control fish that were mock-infected varied little throughout the experiment and little at early timepoints relative to the non-IS groups. When compared to this mock-infected group, the three IS groups were completely depleted of IgM + B cells . Lymphopoiesis only recovered and produced homeostatic B cell counts on day 41 post-infection in some groups and some individuals . In contrast, the immunosufficient group had circulating B cells and this compartment began expanding as early as day 21 post- S. molnari infection. At this point, the number of peripheral blood IgM + B cells increased gradually starting with the group that received the highest dose of 2,500,000 parasites followed by the group receiving ten times less, until IgM + B cell counts were four-fold that of the control group on day 41 in the 6 th week of infection, the timepoint when we previously observed a peak of B cell expansion ( 26 ). The dose-dependent expansion of the B cells may be a sign of B cell activation and an antigen-specific response. Surprisingly, the blood B cell concentration of the group infected with 25,000 BSs was not significantly different from the mock-infected group . Section title: The B cell compartment expands and produces S. molnari -specific antibodies that limit parasitemia Educational score: 4.139134407043457 Domain: biomedical Document type: Study Language: en To measure the B cell response and the outcome of the infection, we quantified antibody titres and parasitemia throughout the experiment. Only baseline non-significantly different anti- S. molnari IgM antibodies were detected in the mock-infected and the IS fish . In immunocompetent fish, specific antibody titres were dose-dependent. Curiously, in the group that received the fewest parasites, antigen-specific IgM titres remained at baseline , with a titre comparable to that of the mock-infected and all IS groups . We measured an increase in anti- S. molnari antibody titres as early as day 28 post-infection in the group receiving the highest dose of parasite (2,500,000 per fish), with the group receiving 10 times fewer parasites following shortly a week later. By the end of the experiment (41 days post-infection), specific antibody titres in these two groups were exponentially higher than in any other group. Overall, these two groups had comparable anti- S. molnari IgM titres but significantly higher titres than any other group . Section title: The B cell compartment expands and produces S. molnari -specific antibodies that limit parasitemia Educational score: 4.153173923492432 Domain: biomedical Document type: Study Language: en In IS fish, an exponential escalation of the infective dose (between 25,000 to 2,500,000) was matched by an exponential dose-dependent increase in parasitemia . In contrast, the measurements of parasitemia in immunosufficient fish varied in kinetics (e.g., the timepoints at which parasitemia peaks). In these groups, the level of BSs in the blood of immunocompetent fish was ultimately dose-independent unlike in IS groups—parasitemia was not significantly different among these groups , and by day 41, all had over 2900-fold less parasitemia than any IS group. Section title: The B cell compartment expands and produces S. molnari -specific antibodies that limit parasitemia Educational score: 4.189974784851074 Domain: biomedical Document type: Study Language: en Taken together, our infection model allows us to study the contribution of the B cells to the anti-parasitic response. The immunocompetent fish receiving the lowest dose of 25,000 S. molnari had neither increased B cell counts nor increased specific antibody titres . Instead, it is possible that they were protected by non-B cells which helped clear the parasite. Overall, we observed that a B cell response requires activation and proliferation, before culminating in antibody secretion. Our data indicate that the immune system of the common carp mounts a protective humoral/B cell response proportional to the parasite inoculum/challenge, with antibodies at least partly suppressing the parasite that would otherwise multiply unchallenged. Section title: In response to S. molnari infection, IgM + B cells proliferate predominantly in the lymphoid tissue, and express gene signatures of activation and differentiation Educational score: 3.4523205757141113 Domain: biomedical Document type: Study Language: en We shifted our attention to the splenic and head/anterior kidney lymphoid tissues where B cell responses are initiated. To characterize the B cell response, we measured proliferation and gene expression as indicators of activation and differentiation. Section title: In response to S. molnari infection, IgM + B cells proliferate predominantly in the lymphoid tissue, and express gene signatures of activation and differentiation Educational score: 4.204825401306152 Domain: biomedical Document type: Study Language: en We detected significantly higher proportions of proliferating IgM + B cells in the blood and head kidney beginning on week 4 . Proliferation uniformly peaked (reached its highest point) and was significantly different from corresponding control groups at week 5 post-infection in all compartments . This preceded the peak in both blood B cell numbers and antibody titres by a week . EdU incorporation and proliferation returned to baseline in all compartments two weeks later, indicating resolution of acute S. molnari infection. As for site specificity, the main compartment of proliferation at week 5 was the head kidney lymphoid organ where over 16% of IgM + B cells had incorporated EdU versus about 7% in the blood and 8% in the spleen . Proliferation being predominantly in the head kidney indicates that it may be a major site of B cell activation and differentiation in S. molnari infection. Section title: In response to S. molnari infection, IgM + B cells proliferate predominantly in the lymphoid tissue, and express gene signatures of activation and differentiation Educational score: 4.378777503967285 Domain: biomedical Document type: Study Language: en To uncover and explain the changes that the B cells are undergoing in the head kidney throughout the infection, we profiled gene expression of magnetic-activated cell sorted (MACS-sorted) IgM + cells . We selected several predicted orthologues of mammalian and fish markers of B cell differentiation and/or survival ( 13 , 34 – 39 ). Among the most differentially expressed genes , the significant downregulation of membIgM (membrane-bound or cell surface IgM), and upregulation of secIgM (secretory IgM), xbp1 , and tnfrsf13b may together indicate differentiation of B cells into antibody-secreting and memory cells . Specifically, the peak of secIgM and xbp1 expression at week 5 post-infection corresponds with the timepoint when we began measuring an increase in anti- S. molnari IgM titres and may be indicative of B cell differentiation into antibody-secreting plasmablasts. The most differentially expressed gene was tnfrsf13b which was a significant 6 log units higher than the control group only at week 9 . As a receptor in the BAFF/APRIL axis that promotes B cell survival and is also upregulated in rainbow trout by myxozoan infection ( 38 , 39 ), expression of tnfrsf13b (alias taci ) may indicate the late differentiation of memory B cells. To date, no one has identified an orthologue of the human plasmablast marker syndecan-1 (alias cd138 ) in teleost fish ( 40 ). We measured expression of the related syndecan-3 but only observed that it was significantly downregulated at weeks 3 and 5. pax5 and cxcr5 expression were unchanged but the former trended toward downregulation at late timepoints. Regarding tissue specificity, a two-way ANOVA determined that secIgM , xbp1 , and tnfrsf13b expression were significantly higher in the head kidney than in the blood compartment. Post hoc testing determined that secIgM (at weeks 5, 7, and 9) and xbp1 (weeks 7, and 9) were significantly overexpressed in the head kidney but not the blood . These late-stage and lymphoid organ-specific changes may indicate that secIgM and xbp1 are markers of B cell differentiation in the head kidney. Section title: In response to S. molnari infection, IgM + B cells proliferate predominantly in the lymphoid tissue, and express gene signatures of activation and differentiation Educational score: 4.112438678741455 Domain: biomedical Document type: Study Language: en We further characterized these specimens via multiplex qPCR by profiling expression of B cell markers that were identified by Pan et al. in Ctenopharyngodon idella grass carp, another cyprinid species ( 16 ). Specifically, they are transcripts the authors identified via single-cell RNA sequencing of sorted head kidney IgM + B cells. Here we present the expression levels of their potential Cyprinus carpio orthologues in our MACS-sorted IgM + B cells . Expression of the 18 genes were hierarchically clustered within the three different compartments throughout the 10-week experiment for comparison between the blood, spleen, and head kidney compartments . Section title: In response to S. molnari infection, IgM + B cells proliferate predominantly in the lymphoid tissue, and express gene signatures of activation and differentiation Educational score: 4.196737289428711 Domain: biomedical Document type: Study Language: en The genes were categorized by their annotation and expected roles according to Pan et al.’s single-cell RNA sequencing data ( 16 ), and/or the cell population they help identify . The expression of cd83 , cxcr4b , egr1 , klf2a , and ier2 were significantly different compared to the non-infected control group and the results of post hoc multiple comparisons tests are summarized in Supplementary Figure S4 . Notably, three of them are activation markers. klf2a , and egr1 were downregulated at early timepoints (specifically, week 2 post-infection); egr1 was later upregulated between week 5 and 8 preferentially in the lymphoid organs while upregulation of klf2a was delayed and centered around week 8 . Among the co-stimulatory molecules, cd83 was significantly overexpressed relative to the control group in all compartments and throughout the experiment with the exception of the week 5 and week 6 timepoint unlike cd86 which peaked at week 1 in all three compartments. Relative to the non-infected control group, cxcr4b was overexpressed at almost all timepoints between week 2 and week 10 in the splenic and head kidney lymphoid organs . c xcr4b may be the functional carp orthologue for retaining or directing select B cells to lymphoid tissues unlike cxcr4a and cxcr5 . Section title: In response to S. molnari infection, IgM + B cells proliferate predominantly in the lymphoid tissue, and express gene signatures of activation and differentiation Educational score: 4.199026107788086 Domain: biomedical Document type: Study Language: en A notable difference with Pan et al.’s study ( 16 ) is that we are studying these markers at the population level (in bulk) rather than at the level of single cells. Therefore, we still expect changes in subpopulations of IgM + B cells and changes in B cell marker expression even if these are more difficult to detect and not statistically significant. Among the markers whose expression was not significantly different from the control group, c d34 is a marker of mouse and human hematopoietic stem cells. cd34 expression was downregulated relative to the control group in all compartments during the first 5 weeks of infection . Two markers of innate B cells, irf4 and irf8 , trended oppositely with the former highly overexpressed and the latter downregulated at early timepoints in all compartments. Among the pan-B cell markers, the most notable change was a 2.5-log upregulation and a 4-log downregulation of igm compared to their controls in the blood and head kidney, respectively, at the same week 3 timepoint. Similarly, the expression patterns of the activation markers mki67 and top2a matched those of igm in the same cellular compartments at the same week 3 timepoint, suggesting that all three may be markers of the same IgM + cell subpopulation. Finally, we again observed a reverse pattern of xbp1 and pax5 expression which is an indicator of an ongoing immune response and B cell differentiation . Section title: In response to S. molnari infection, IgM + B cells proliferate predominantly in the lymphoid tissue, and express gene signatures of activation and differentiation Educational score: 4.159241676330566 Domain: biomedical Document type: Study Language: en In summary, considering that all these markers define distinct cyprinid IgM + B cell subpopulations ( 16 ), our results suggest that there is: reduced hematopoiesis or lymphopoiesis; activation of an innate B cell population; antigen-presenting cell (APC) activity or costimulation; activation, proliferation, and differentiation of B cells; migration/retention of cells in lymphoid tissues. Together, these changes may be the origin of the humoral response against the parasite . Section title: Memory B cells are produced throughout the acute response to S. molnari and persist as resting cells Educational score: 3.9963715076446533 Domain: biomedical Document type: Study Language: en Fish that survive a primary myxozoan infection presumably harbor memory lymphocytes that protect them from secondary infections. We gathered evidence that the initial immune response produces a greater breadth of B cells than we could previously appreciate with anti-carp IgM staining alone. Yet, we could not directly identify memory cells as there is so far no marker for let alone a defined subpopulation of teleost memory B cells. Furthermore, we must study these cells on the order or scale of months that are most relevant to immunological memory, vaccine success, and protection from natural seasonal (re-)infections. Section title: Memory B cells are produced throughout the acute response to S. molnari and persist as resting cells Educational score: 4.105288982391357 Domain: biomedical Document type: Study Language: en We revisited the EdU pulse labeling method, and searched for EdU + IgM + B cells months after infection which should theoretically reveal B cells that were i) activated upon encountering S. molnari (antigen), ii) proliferated and incorporated EdU as a result, and iii) differentiated into long-lived quiescent cells that retain the EdU. The latter are selected into the memory pool following the contraction and resolution phases of the primary immune response. To test this, we assigned fish to six labeling windows/groups receiving EdU during a specific week following acute S. molnari infection. Approximately six months after infection and approximately five months following EdU injection, we detected EdU + cells in the blood, spleen, and head kidney compartments . Section title: Memory B cells are produced throughout the acute response to S. molnari and persist as resting cells Educational score: 4.178780555725098 Domain: biomedical Document type: Study Language: en Astonishingly, EdU + IgM + B cells were readily detectable in the lymphocyte gate, representing around 2% to 5% of all IgM + B cells and potentially include memory cells. In contrast, a negligible amount of weakly EdU + cells were detected among granulocytes from the same fish . Compared to proliferation during the acute infection , the distribution of EdU + cells was mainly in the blood and spleen rather than the head kidney, which matches the location of the resting B cells described by Ma et al. ( 13 ); interestingly, they were not labeled during the 5 th week of acute infection, and peak proliferation . The majority of EdU + IgM + cells originated from week 7 post-infection and at the time of measurement, represented 6% of all splenic IgM + cells, and this number was about 4% and 3% in the peripheral blood and head kidney, respectively. Section title: Memory B cells are produced throughout the acute response to S. molnari and persist as resting cells Educational score: 4.04624080657959 Domain: biomedical Document type: Study Language: en In other words, out of all the proliferating and EdU-incorporating cells during acute infection, a subset survived, having not been turned over, not proliferated further, and not diluted EdU below the threshold of detection. This B cell population may be partly composed of the fish equivalent of the memory B cell, a non-dividing recirculating B cell subset awaiting reactivation. Section title: A head kidney-exclusive B cell subpopulation with high levels of intracellular IgM may represent plasma cells Educational score: 4.128665447235107 Domain: biomedical Document type: Study Language: en Plasma cells and their antibody effector molecules make up another pillar or wall of humoral memory. We were able to detect anti- S. molnari antibodies eight months post-infection, long after resolution of parasite infection and without any reinfection or immunization . With these antibodies being presumably produced by plasma cells, we devised methods to detect this B cell subset based on the exceptional amounts of Ig they produce, their levels of membrane IgM (memb IgM) ( 36 ), their size, their compartmentalization in the head kidney ( 13 ), and hypothesized resistance to hormonal stress ( 41 ). Section title: A head kidney-exclusive B cell subpopulation with high levels of intracellular IgM may represent plasma cells Educational score: 4.170295715332031 Domain: biomedical Document type: Study Language: en Thus, we costained cells in a step-wise manner to detect the extracellular membrane IgM (Memb IgM) and intracellular IgM (IC IgM) using a single monoclonal anti-carp IgM antibody. In the blood and spleen, the B cells stained minimally for IC IgM with a slight upward shift of the BCR high population of cells . This did not reveal any new population among the cells we have always detected . However, the head kidney revealed two new B cell populations that were previously undetectable through Memb IgM staining alone : a Memb IgM - IC IgM mid population and a Memb IgM mid IC IgM high population. The former is relatively abundant (representing around 3% of all leukocytes) while the latter is exceedingly rare (representing around 0.5% of all leukocytes). Overall, based on their phenotype and tissue-specificity, we hypothesized that one of these populations represents plasma cells. Section title: A head kidney-exclusive B cell subpopulation with high levels of intracellular IgM may represent plasma cells Educational score: 4.185686111450195 Domain: biomedical Document type: Study Language: en To test these cells’ resistance to stress, we immunosuppressed fish that had previously been infected with S. molnari . As corticosteroid-induced immunosuppression targets lymphopoiesis ( 42 ), dividing cells, and short-lived cells, we hypothesized that plasma cells would be resistant and would not be depleted unlike their naïve B cell counterparts. This treatment eliminates virtually all IgM + B cells from the blood . In the spleen and head kidney, this approach depleted the most abundant B cell populations including the Memb IgM - B cell population . In contrast, the Memb IgM mid IC IgM high population became readily detectable in the head kidney (over 2% of all leukocytes in the example). We also hypothesized that they would continue producing antibodies despite the immunosuppression and were responsible for keeping the parasite latent long-term. Thus, we measured antibody titres and parasitemia in the weeks following immunosuppression . Anti- S. molnari IgM antibody titres doubled and coincided with the reemergence of the parasite and an over 10-log increase in parasitemia at the final timepoint of 41 days post-immunosuppression. Section title: A head kidney-exclusive B cell subpopulation with high levels of intracellular IgM may represent plasma cells Educational score: 4.15897274017334 Domain: biomedical Document type: Study Language: en Together, our data indicate that the initial B cell response elicits constitutive antigen-specific antibody production. The source of these antibodies may be the corticosteroid-resistant, head kidney-resident, IC IgM high B cell population, which are significantly larger and denser than their counterparts with high Memb IgM expression . These cells and antibodies may be one component of constitutive defense keeping S. molnari latent and/or protecting the host from future infections. Section title: Discussion Educational score: 4.078198432922363 Domain: biomedical Document type: Study Language: en Overall, our data indicate that B cell activation follows a similar course to that in ‘higher’ vertebrates and produces specialized cellular subsets with memory and (constitutive) antibody-secreting phenotypes, together forming ‘two walls of protection’ ( 43 ). Our results indicate that the initial B cell response is protective and prevents the worst form of disease caused by S. molnari (the only two deaths recorded in this experiment were from the IS groups). Section title: The B cell response, memory B and plasma cells in a teleost fish Educational score: 4.074220657348633 Domain: biomedical Document type: Study Language: en In mammals, antigen activates B cells and they differentiate in lymphoid tissues. This process can be divided into at least two phases: an initial phase that produces the short-lived plasmablasts and some memory B cells, whereas a second phase produces memory B and long-lived plasma cells that have affinity-matured and have been positively selected for in germinal center reactions ( 44 , 45 ). Section title: The B cell response, memory B and plasma cells in a teleost fish Educational score: 4.179503917694092 Domain: biomedical Document type: Study Language: en The B cell response we observed was dose-dependent, and antigen-specific. In comparison to the response of rainbow trout to the model hapten-protein antigen TNP-KLH, anti- S. molnari antibody production was accelerated, and titres were exponentially higher at the same timepoint ( 12 ). The initial surge in antibody production at the 5-week timepoint is likely the product of short-lived plasma cells or plasmablasts and matches the timing of the significant increase in igm transcripts we previously observed in the same model and in this study ( 26 ). Section title: The B cell response, memory B and plasma cells in a teleost fish Educational score: 4.288278102874756 Domain: biomedical Document type: Study Language: en We measured a peak of proliferation at week 5 post-infection and detected a significant proportion of EdU + IgM + B cells in both the head kidney and spleen. These cells likely include those described in a recent report by Shibasaki et al. of a germinal center analogue in the rainbow trout spleen ( 33 ). By detecting highly proliferating B and T cells adjacent to melanomacrophages, Shibasaki et al. convincingly identified sites that support clonal expansion, antigen receptor affinity maturation, and clonal selection. One expected output is antibody-secreting cells which circulate and contribute to the sharp rise in the number of IgM + and EdU + B cells at week 5 and 6 in the blood even though it is not a site of proliferation . The short-term distribution of antibody-secreting cells throughout the periphery was also observed by Davidson et al. in Limanda limanda following immunization with human gamma globulin ( 11 ). The precise identity of the circulating B cells can be confirmed with a strategy like the one we used to detect the kidney-resident IC IgM high cells. However, they are only expected to be short-lived and provide short-term protection. Section title: The B cell response, memory B and plasma cells in a teleost fish Educational score: 4.261462688446045 Domain: biomedical Document type: Study Language: en Regardless of which phenotypic markers they express, regardless of species-to-species variations, the purest and most universal definition of what is a memory B cell is their longevity. We traced the proliferating cells of the primary response to determine if they persist long after resolution of the acute response. We hypothesize that the EdU + cells we detected months after infection/labeling include clones that avoided caspase-mediated apoptosis in the melanomacrophage centers ( 33 ). The upregulation of secIgM , xbp1 , tnfrsf13b , and downregulation of membIgM , and pax5 may also be byproducts of ongoing selection and differentiation. We detected memory B cells in every compartment, and they formed at every timepoint studied. The memory B cells detected in the spleen and blood matches where Ma et al. observed them in rainbow trout ( 13 ). These cells are likely recirculating between the two compartments, explaining why their proportions are very similar in the two compartments. In contrast, the head kidney of carp and other bony fish species appears to be a niche for plasma cells that do not recirculate. These cells likely reside in the head kidney which is a survival niche for plasma cells of other bony fish species as well ( 13 , 14 , 16 , 46 ). Section title: The B cell response, memory B and plasma cells in a teleost fish Educational score: 4.1978044509887695 Domain: biomedical Document type: Study Language: en The emergence of memory cells and the peak of their formation in week 7 was relatively late and did not correlate with the peak of B cell proliferation in week 5 . Considering the timing and selection mechanisms ongoing in lymphoid tissues, it is likely that early memory B cells express low affinity BCRs and this affinity gradually matures as observed in rainbow trout and channel catfish ( 12 , 13 , 15 ) driven by activation-induced cytidine deaminase-mediated antigen receptor diversification in germinal center analogues ( 33 ). In our study, we hypothesize that the late-stage memory cells emerging in week 7 may be successful progenitors of melanomacrophage center reactions, and express affinity-matured BCRs which confer a survival/fitness advantage. Section title: Humoral memory and the implications for vaccination and natural immunity Educational score: 4.219328880310059 Domain: biomedical Document type: Study Language: en In humans, there is evidence that exposure to a pathogen is sufficient to protect an individual for a lifetime: in a 2008 study, survivors of the 1918 H1N1 influenza pandemic continued to harbor memory cells producing strain-specific anti-hemagglutinin neutralizing antibodies, nearly 90 years after exposure to the virus ( 47 ). If fish B cells behave in much the same way, there are major implications for vaccination and natural acquired immunity against pathogens. In a study similar to ours in mice, memory B and plasma cell retained BrdU at the 8-week timepoint ( 48 ). Here, we continued to detect EdU + , IgM + B cells well after 6 months post-infection and EdU pulse labeling. A recent Atlantic salmon vaccine trial demonstrated persistent antigen-specific antibody titres over 80 weeks post-immunization, in a full aquaculture production cycle ( 18 ). Gilthead seabream that had survived a previous infection with the myxozoan Enteromyxum leei maintained specific antibody titres for over 16 months ( 17 ). This progress and such milestones are consequential because we need to know how long memory is maintained post-vaccination or whether fish having already survived an initial infection will be immune during next year’s outbreak. Understanding how this memory is generated and maintained will help us move away from purely empirical vaccines and methods, and towards conferring acquired immunity. Section title: Humoral memory and the implications for vaccination and natural immunity Educational score: 4.309305191040039 Domain: biomedical Document type: Study Language: en Existing models propose that memory B cells could be maintained by continuous antigen exposure while others propose that their longevity may be intrinsic to ‘tonic’ BCR signaling ( 44 ). The aforementioned Atlantic salmon vaccine trial demonstrated retention of select antigens many months post-vaccination in granulomas forming in the pancreas but not in lymphoid organs ( 18 ). Alternatively, B cells could also be re-exposed to antigen via booster immunizations to provide survival signals and maintain antibody titres ( 44 ). Considering the discovery of a germinal center analogue in fish ( 33 ), the fish memory B cells we detected may potentially also re-enter melanomacrophage centers to further diversify their antibody repertoire and/or differentiate into (short-lived) plasma cells, providing a ‘faster’ and ‘stronger’ secondary immune response as occurs in mammals ( 44 ). Section title: Humoral memory and the implications for vaccination and natural immunity Educational score: 4.27105712890625 Domain: biomedical Document type: Study Language: en Finally, we do not know if the survival of memory cells in laboratory settings would be reproducible in natural settings where animals are exposed to environmental stressors. At least in Atlantic salmon in a farm setting, specific antibody titres were maintained over 80 weeks post-immunization despite the seasonal changes in temperature and photoperiod ( 18 ). In our study, we mimicked the effects of stress-induced cortisol/glucocorticoids in S. molnari -infected fish. Abnormally high stress-induced plasma cortisol increases the susceptibility of trout to fungal and bacterial infection ( 49 ). In our study, after administration of the corticosteroid, antibody titres remained stable without de novo B cell lymphopoiesis ( 44 ), without circulating naïve B cells, and without de novo antibody production, despite the half-life of fish IgM being reportedly mere days ( 50 ). Thus, it suggests that anti- S. molnari plasma cells would survive such a stressor and that their antibody production is not affected . The resistance of plasma cells may also be non-hormonal as observed in rainbow trout plasma cells resistant to hydroxyurea ( 14 ). The overall resilience of plasma cells may make them key to long-term protection that supports, for example, the physiology and life cycle of spawning fish as hypothesized by Zwollo ( 41 ). In this ‘immunological imprinting’ scenario, plasma cells are generated in juvenile fish against pathogens in their rearing site. As adults returning to these sites during their ‘spawning journey’, Zwollo hypothesizes that plasma cells may be resistant to cortisol, have a survival advantage over other B cell populations, and continue producing antibodies in anticipation of familiar pathogens ( 41 ). Section title: Humoral memory and the implications for vaccination and natural immunity Educational score: 4.125912666320801 Domain: biomedical Document type: Study Language: en Mechanistically, fish splenic and kidney leukocytes express glucocorticoid receptors ( 51 ) and cortisol induces apoptosis of C. carpio peripheral blood leukocytes ( 52 ). In addition to higher bacterial disease susceptibility ( 49 ), another study using triamcinolone acetonide demonstrated that it can nullify immune protection and perhaps memory of C. carpio against the protozoan parasite Ichthyophthirius multifiliis via an unknown mechanism ( 31 ). Interestingly, the same authors measured unchanged specific antibody titres following administration of the drug ( 30 ). Section title: Potential lessons from the S. molnari infection model Educational score: 4.083082675933838 Domain: biomedical Document type: Study Language: en Chronic infections pose the greatest challenges even in well-studied species. For example, despite decades of concentrated research efforts, there is still no vaccine available against pathogens causing chronic diseases such as human immunodeficiency virus and malaria. Interestingly, in malaria infection, despite robust production of plasmablasts and anti- Plasmodium antibodies, this was ultimately metabolically taxing and impaired production of memory B and plasma cells ( 53 ). Our data suggests that we can rule out this possibility to explain chronic S. molnari infection because we measured a persistent constitutive anti- S. molnari response. Section title: Potential lessons from the S. molnari infection model Educational score: 4.374588966369629 Domain: biomedical Document type: Study Language: en In natural settings, could immunosuppression (e.g., via cortisol) be a trigger for parasite activation, spore formation, and perpetuating the life cycle? Sterilizing immunity to myxozoan infections is rare and fish become carriers, suggesting that the parasite can evade the immune system, potentially a general feature of myxozoans that have co-evolved with fish hosts ( 54 , 55 ). Despite continued secretion of anti- S. molnari antibodies, humoral memory could not provide sterilizing immunity in chronically infected fish . Even though the immune antisera of infected fish can lyse the parasite (manuscript in preparation), the latent parasite was able to re-emerge 41 days after administration of the corticosteroid. It is possible that plasma cells expressing antibodies specific to an original ‘founder’ variant could no longer recognize an escape variant of the parasite, or that other immune non-B cells are protective and also eliminated by the corticosteroid we administered, i.e., the B cell response is necessary but insufficient alone. Taken together, myxozoan parasite escape and sporulation may be side effects of the ‘immunological imprinting hypothesis’ and hormonal immunosuppression ( 41 ). In this model, interseasonal carriers such as the common carp or wild trout may spawn and produce invertebrate-infective spores. Infected invertebrates may produce the fish-infective spores, completing the parasite life cycle and causing the annual outbreaks of myxozoan infections. Section title: Concluding remarks Educational score: 3.8576459884643555 Domain: biomedical Document type: Study Language: en In summary, we adapted and devised methods that may be applicable to other vertebrate species in which adaptive immunity and immunological memory can be demonstrated because it relies on a common phenomenon, not a common mechanism or marker. Perhaps what holds true for all vertebrates and the only all-encompassing definition is that memory lymphocytes are antigen-experienced after engaging in a past immune response, and persist after resolution of that response. Immunologists have observed immunological memory for over a century and achieving humoral memory will help us meet the needs of immunologists, parasitologists, and fish husbandry. Section title: Key resources table Educational score: 1.663114070892334 Domain: biomedical Document type: Study Language: en Please refer to Table 1 for the key resources used in this study. Section title: Experimental model details Educational score: 1.1080435514450073 Domain: biomedical Document type: Other Language: en Animal procedures were performed in accordance with Czech legislation . Animal handling complied with the relevant European guidelines on animal welfare and the recommendations of the Federation of Laboratory Animal Science Associations. The animal experiments were approved by the Ministry of Education. Approval ID: MSMT-4186/2018-2. Our study is reported in accordance with ARRIVE guidelines ( https://arriveguidelines.org ). Section title: Experimental model details Educational score: 4.1093950271606445 Domain: biomedical Document type: Study Language: en We reared specific pathogen-free (SPF) common carp ( Cyprinus carpio ) from peroxide-treated fertilized eggs (700 mg/L for 15 min) in an experimental recirculating system in the animal facility of the Institute of Parasitology, Biology Centre CAS. During the experiment, fish with an average mass of approximately 25 g were selected and fed twice a day with a commercial carp diet (Skretting) at a daily rate of 1.5% of their body mass. The sex of experimental fish was not considered in our study. The fish were implanted with passive integrated transponders to track the experimental group they belonged to. We housed fish randomly and therefore fish from different experimental groups shared the same tanks. However, all tanks shared the same water which was UV-irradiated, ozonized water at 21 ± 1°C, with water quality (oxygen, pH, ammonia, nitrite and nitrates) monitored daily using probes and titration tests. Ammonia levels never surpassed 0.02 mg/L. Section title: Experimental infections and fish Educational score: 3.5531396865844727 Domain: biomedical Document type: Study Language: en For all infections, we collected S. molnari parasite from the fish host and purified the BSs via DEAE cellulose (BIOpHORETICS, Sparks, NV, USA) isolation as described previously ( 19 ). Section title: Experimental infections and fish Educational score: 4.129078388214111 Domain: biomedical Document type: Study Language: en For the trial measuring B cell counts, antibody titres, and parasitemia, fish were divided into eight groups of six fish each for the initial kinetic measurements of peripheral blood concentration, antibody titres, and parasitemia: groups 1-3 were immunocompetent/immunosufficient and received one of three ten-fold diluted doses of parasite ranging from 2,500,000 to 25,000; groups 4-6 were also given one of the three doses of parasite as described for groups 1-3 but these fish were immunosuppressed with triamcinolone acetonide (Glentham Life Sciences, Corsam, UK) at a dose of 200 µg/g of body mass the day before intraperitoneal injection of the parasite ( 19 , 30 – 32 , 56 ); finally, one group was mock-infected via injection of sterile Roswell Park Memorial Institute 1640 medium in the same site . RPMI 1640 also served as the medium for ex vivo parasite. Fish that were not immunosuppressed were instead mock-treated with sterile PBS, the diluent used for the triamcinolone acetonide. Section title: Experimental infections and fish Educational score: 3.9283998012542725 Domain: biomedical Document type: Study Language: en For the downstream EdU labeling assay, we infected two additional groups of fish with 50,000 BSs per fish, and mock-infected one more group. One group was mock-infected with RPMI 1640 (n = 27) and served as a control for the second group infected with S. molnari (n = 36). The final third group was divided into six sub-groups (n = 4 each) for the purpose of memory B cell detection. Section title: Experimental infections and fish Educational score: 3.753674268722534 Domain: biomedical Document type: Study Language: en To measure differential gene expression, 50 fish were infected with one million BSs each, five fish for each of ten sampling timepoints, sampled at one-week intervals for the ten-week duration of the experiment. An additional five non-infected fish were dissected immediately prior to commencing the infection trial, and they served as the control group. Section title: Experimental infections and fish Educational score: 3.2829272747039795 Domain: biomedical Document type: Study Language: en A final independent cohort was infected with 50,000 BSs per fish before immunosuppression rather than immunosuppressed first. We subjected this group to a 223-day gap between infection and treatment with triamcinolone acetonide (as described above) to study immunological memory. Section title: Measuring peripheral blood B cell concentration Educational score: 4.1213836669921875 Domain: biomedical Document type: Study Language: en We collected minimal quantities (around 50 to 100 μL) of blood from experimental fish at a frequency of twice weekly throughout the 6-week experiment. The blood was collected with 30-gauge needles into heparinized syringes (via rinsing of the needle and syringe with 200-300 μL of 5,000 IU/mL heparin sodium before use). In preparation for flow cytometry, 4 μL of the whole blood specimens were aliquoted, the blood was resuspended in 200 μL of RPMI 1640 before 100 μL was discarded. Preparation of specimens for flow cytometry was in the 96-well plate format. We centrifuged the cells at 500 g for 3 min and washed them once more to dilute and remove any serum (all washes and stains were in the absence of antibiotics or bovine serum). In parallel, 2 μL of the whole blood was collected in TNES-urea to measure parasitemia. The remainder of the heparinized blood was centrifuged at 500 g for 5 min. We collected and froze the plasma at -20°C until necessary for downstream applications. Section title: Measuring peripheral blood B cell concentration Educational score: 4.161019325256348 Domain: biomedical Document type: Study Language: en For flow cytometry, the cells were then stained with previously titrated SP2/0 hybridoma supernatant containing the monoclonal anti-carp IgM antibody (clone WCI12) ( 57 ) diluted 1:100 in RPMI 1640. We incubated cells for 20 min in ice before subjecting cells to two washes to remove excess antibodies. 1:500 goat anti-mouse IgG1-APC (Abcam, Cambridge, UK) was then applied as a secondary detection reagent, also diluted in RPMI 1640. Incubation was for 20 min in ice before we spun cells down, and added 3,3’-dihexyloxacarbocyanine iodide [DiOC6(3)] (Life Technologies, Carlsbad, CA, USA) diluted to 5 μg/mL in RPMI 1640. We used this lipophilic dye as an alternative to any density centrifugation step, to distinguish the relatively metabolically active and endoplasmic reticulum- or mitochondria-rich leukocytes from the relatively inactive red blood cells. All stains were performed in a volume of 100 μL. A final centrifugation took place before we resuspended cells in a final volume of 200 μL of RPMI 1640. The cells were measured on the BD FACSCanto II (BD Biosciences, Prague, Czech Republic) and data was recorded for 20 s at a medium flow rate (60 μL/min) for all samples, allowing us to quantify both proportion and absolute numbers of B cells in the original 2 μL of whole blood. Section title: Measuring peripheral blood B cell concentration Educational score: 3.1322407722473145 Domain: biomedical Document type: Study Language: en We used a two-laser configuration of the BD FACSCanto II: equipped with a 488-nm blue laser and a 633-nm red laser. Section title: Enzyme-linked immunosorbent assay (ELISA) measurement of relative antibody titre Educational score: 4.097134113311768 Domain: biomedical Document type: Study Language: en We designed an indirect ELISA and calculated the relative antibody titre from each fish plasma sample according to a method established by Sacks et al. ( 58 ). Our objective was to quantify the amount of S. molnari -specific IgM in fish plasma and hence the antigen coating remained constant throughout the experiment. We collected and pooled together parasite lysate derived from three different individual fish at a 1:1:1 mass ratio. Live parasite BSs were pelleted and resuspended in phosphate-buffered saline (PBS) supplemented with the cOmplete Protease Inhibitor Cocktail (Roche Diagnostics GmbH, Mannheim, Germany) by adding one part (volume-wise) of the manufacturer’s recommended stock solution to 24 parts of PBS. This suspension was subjected to a minimum of three freeze-thaw cycles before the lysate was centrifuged at 13,000 g for 10 min at 4°C. We collected the supernatant and measured the absorbance at 280 nm to estimate the protein content using a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA). This exact antigen pool was aliquoted and frozen until all plasma samples were collected for the ELISA. Section title: Enzyme-linked immunosorbent assay (ELISA) measurement of relative antibody titre Educational score: 4.230869293212891 Domain: biomedical Document type: Study Language: en The S. molnari antigen was diluted to 25 μg/mL in 29 mM Na 2 CO 3 71 mM NaHCO 3 pH 9.6 coating buffer. 100 μL were added to wells of a 96-well flat-bottom immunoGrade plates (Carl Roth GmbH, Karlsruhe, Germany) and left to incubate overnight at 4°C. The coating solution was decanted the next day and 100 μL of PBS 5% non-fat dry milk powder (5 g/100 mL) was added to each well. We left the plate blocking at ambient temperature for 3 hours. We washed away the blocking buffer three times using 100 μL of PBS 0.1% Tween-20. We then added either 50 μL of PBS (for background subtraction) or experimental fish plasma diluted in PBS in triplicate. The specific details of fish plasma dilution and which samples to include on each plate are explained by Sacks et al. ( 58 ). Briefly, a dilution series of a seropositive fish plasma from another experiment served as a standard and was included on every plate ( 59 ). We measured the specific antibody titre of this specimen in an independent experiment through a two-fold plasma dilution series. The last dilution with an absorbance above the mean absorbance plus two SDs of a seronegative SPF naïve cohort (n = 5, plasma diluted 1:100) was considered the antibody titre of the standard. We ensured that we had enough of this plasma for every plate we intended to run onwards. Thus, a two-fold dilution series of the standard, ranging from 1:200 to 1:12,800, was included on every plate for the purpose of determining the relative antibody titre and to account for inter-assay and -plate variation. The experimental fish plasma was generally diluted either 1:100 for the naïve group or 1:200 for the infected group, provided the absorbance at 450 nm (A450nm) fell within the range of the standard curve. Otherwise, a specimen was assayed again at a higher or lower dilution as appropriate. The plasma was incubated for 1 hour at ambient temperature. It was washed away thrice with PBS 0.1% Tween-20. We repeated these incubation steps sequentially with a 100 μL of secondary mouse anti-carp IgM antibody and a tertiary anti-mouse IgG-horse radish peroxidase conjugate antibody diluted 1:5000 (Sigma-Aldrich, Darmstadt, Germany) before a final wash step (five washes) and signal development by adding 100 μL of pre-warmed TMB per well (Thermo Fisher Scientific, Rockford, IL, USA). The plates were incubated for 6 min before we stopped the reaction with 2 M sulfuric acid. Section title: Enzyme-linked immunosorbent assay (ELISA) measurement of relative antibody titre Educational score: 3.8098323345184326 Domain: biomedical Document type: Study Language: en We measured the A450nm on the Infinite 200 PRO microplate reader (Tecan, Männedorf, Switzerland). Before analysis, all measurements were background-corrected by subtracting the mean of the triplicate PBS internal negative control measurements included on every plate. Section title: qPCR quantification of parasitemia Educational score: 4.1701765060424805 Domain: biomedical Document type: Study Language: en We extracted total DNA from 2 μL of whole blood. The blood was immediately stored in 400 μL of TNES-urea (10 mM Tris-HCl, 125 mM NaCl, 10 mM EDTA, 0.5% SDS, and 4 M urea, pH 8.0) at 4°C. Once the entirety of samples for the experiment had been collected, the DNA was purified via a modified phenol-chloroform extraction as described by Holzer et al. ( 60 ). We measured the yield and the purity via spectrophotometry (ratio of absorbance at 260 nm to 280 nm) using the NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA). All samples were diluted to 100 ng/μL to normalize the input material for the qPCR. We measured relative parasitemia as the quantity of S. molnari SSU rDNA relative to C. carpio β-actin DNA using the primers and probes in a duplex TaqMan real-time PCR assay as described previously ( 26 ). Naturally, severe disease would both increase parasitemia and deplete host erythrocytes ( 32 ), increasing the parasite signal while decreasing the host signal. In all instances and timepoints for the mock-infected group, and for some of the earlier timepoints in infected groups, no parasite was detectable, no PCR product was amplified, and no Ct value was measurable. For these specimens, to enable relative quantification, we assigned them a Ct value of 50, the number of PCR cycles we programmed. Instead of omitting these specimens, we could include them in analyses with Ct values measured from infected specimens. Measurements were made in technical duplicates. The TaqMan qPCR was performed on the QuantStudio 6 (Applied Biosystems, Foster City, CA, USA). Section title: EdU pulse labeling of proliferating and memory B cells Educational score: 3.982602119445801 Domain: biomedical Document type: Study Language: en Experimental fish were injected with the thymidine analogue EdU to measure cells proliferating in vivo during the acute response to S. molnari . The fish in these two groups were each injected intraperitoneally with 100 μL of 5 mM EdU one day before sampling. We sampled four infected and three mock-infected fish per week at one-week intervals between weeks 1 and 9 post-infection. Section title: EdU pulse labeling of proliferating and memory B cells Educational score: 3.768545389175415 Domain: biomedical Document type: Study Language: en A separate group of fish was instead injected three times (at two-day intervals) during a single labeling window/week between weeks 3 and 8 of this experiment. The purpose was to capture the long-term outcome of the proliferation and EdU incorporation events that had occurred during a particular labeling window between different organs and between different windows. Therefore, we sampled these fish approximately six months after each respective labeling window. Section title: EdU pulse labeling of proliferating and memory B cells Educational score: 4.142935276031494 Domain: biomedical Document type: Study Language: en The entirety of the head kidney and spleen were biopsied. We placed the tissues upon the surfaces of 100-μm cell strainers (Corning, Durham, NC, USA). The tissues were then gently dissociated using the textured end of a syringe plunger with simultaneous washing and rinsing with RPMI 1640. The cell suspensions were then loaded onto 25% Percoll (Cytiva, Uppsala, Sweden), prepared with one part Percoll and three parts RPMI 1640. The heparinized blood was loaded onto Ficoll-Paque (Cytiva, Uppsala, Sweden). Leukocytes were enriched by density centrifugation at 500 g for 20 min at 4°C with minimum acceleration and braking. We collected either the pellet for splenocytes and head kidney leukocytes, or the buffy coat for blood mononuclear cells. These cell fractions were washed twice in RPMI 1640. We enumerated the cells by Trypan Blue exclusion in a Bürker chamber. One million were allotted per fish per compartment, they were stained for detection of IgM + cells as described for blood leukocytes in section 4.4 minus the DiOC6(3) staining. Section title: EdU pulse labeling of proliferating and memory B cells Educational score: 4.088534355163574 Domain: biomedical Document type: Study Language: en Cells that had incorporated EdU were detected using the Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Life Technologies Limited, Paisley, UK) according to the manufacturer’s instructions except that the entire protocol was conducted on 96-well non-tissue culture-treated polystyrene V-bottom plates (Greiner Bio-One, Les Ulis, France) with the maximum wash volumes adjusted accordingly: we used 10% of the recommended freshly prepared Click-iT cocktail per specimen, and hence resuspended the cells in only 10 µL of saponin-based permeabilization and wash reagent in the step prior to the Click-iT reaction. Specimens were analyzed on the BD FACSCanto II. Section title: Primer selection Educational score: 3.963191270828247 Domain: biomedical Document type: Study Language: en For SYBR Green-based qPCR, the igm -specific primer pairs (against both membIgM and secIgM ) and housekeeping bactin -specific primer pairs were previously described ( 26 ). We designed the other hypothetical/predicted markers of B cell activation/differentiation and they are described in Supplementary Table S1 . Section title: Primer selection Educational score: 4.142784118652344 Domain: biomedical Document type: Study Language: en For the multiplex qPCR, the gene/primer selection was based on a single-cell RNA sequencing B cell atlas published for grass carp Ctenopharyngodon Idella ( 16 ). The nucleotide sequences of the B cell-specific target genes in common carp were derived from the respective NCBI nucleotide accession using the Pyrosequencing assay design software (Biotage AB, Uppsala, Sweden). For each of the 18 target genes, we designed either the sense or the antisense primer to bind an exon - exon junction. They are outlined in Supplementary Table S1 . eef1a1 (eukaryotic translation elongation factor 1 alpha 1) and rps11 (ribosomal protein S11) were included as reference genes ( 61 ). Section title: Quantitative reverse transcription PCR gene expression profiling of B cell activation and differentiation markers Educational score: 4.21361780166626 Domain: biomedical Document type: Study Language: en Fish were dissected and the B cells stained with monoclonal anti-carp IgM antibody (clone WCI12) as described for the proliferation assay. MACS was performed according to the manufacturer’s instructions using Anti-Mouse IgG Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were stored in RNAlater (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) overnight, centrifuged the next day at 5,000 g either undiluted or diluted 1:1 with PBS. We then stored the pellet at -20°C for RNA isolation using the NucleoSpin RNA kit (MACHEREY-NAGEL, Düren, Germany) according to the manufacturer’s instructions. RNA was aliquoted for long-term storage at -80°C or to prepare a 4 ng/µL solution from which 2.5 µL was used (10 ng) in the 10 µL reaction format of the Power SYBR Green RNA-to-CT 1-Step Kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) as recommended by the manufacturer. Enough of an equally proportioned pool of 12 randomly selected RNA specimens was prepared and assayed as described above as an inter-run calibrator. We made technical duplicate measurements on the QuantStudio 6 (Applied Biosystems, Foster City, CA, USA). We repeated measurements for any technical duplicates that varied over 0.5 cycles. In this case, Ct values were adjusted based on the inter-run calibrator but otherwise, we followed the sample-maximization method for plate/assay design. We analyzed the data via the 2 −ΔΔCT method: all data were initially normalized to the Ct values of bactin from the same RNA specimen (ΔCt), these data were further normalized to the mean of the ΔCt of the corresponding control group (ΔΔCt). Section title: Multiplex qPCR gene expression profiling Educational score: 4.123948574066162 Domain: biomedical Document type: Study Language: en RNA specimens extracted for one-step RT-qPCR described above were also aliquoted and reverse-transcribed into cDNA using the Reverse Transcription Master Mix (Standard BioTools, San Francisco, CA, USA) in a Thermocycler-T1 (Biometra, Analytik Jena, Jena, Germany) instrument. The resulting cDNA was pre-amplifed using the Preamp Master Mix (Standard BioTools) and a primer master mix with a final concentration of 100 µM per primer pair. Finally, preamplified cDNA was treated with exonuclease I (New England BioLabs, Ipswich, MA, USA) and diluted in SsoFast EvaGreen Supermix with Low ROX (Bio-Rad) and 192.24 DELTAgene Sample Reagent (Standard BioTools). Section title: Multiplex qPCR gene expression profiling Educational score: 4.1002397537231445 Domain: biomedical Document type: Study Language: en We used the BioMark HD system (Standard BioTools) to profile the expression of selected B cell marker genes and two reference genes ( Supplementary Table S1 ) in the preamplified cDNA samples. To this end, the primers and pre-amplified cDNAs were inserted to the assay and sample inlets, respectively, on a 192.24 Gene Expression biochip (Standard BioTools). The Control Line Fluid and the Actuation and Pressure Fluid (Standard BioTools) were transferred to the wells provided on the biochip. Having underwent the Load Mix script using the IFC Controller RX (Standard BioTools), the 192.24 Gene-Expression biochip was finally transferred to the BioMark HD-System (Standard BioTools) to perform the quantification reactions. Section title: Multiplex qPCR gene expression profiling Educational score: 4.072918891906738 Domain: biomedical Document type: Study Language: en The Ct values were retrieved using the Fluidigm RealTime PCR Analysis Software v4.5.2 and translated into copy numbers based on external amplicon-specific standard curves ( R 2 > 0.99). The relative copy numbers were normalised by the geometric mean of the expression values of the suitable reference genes eef1a1 and rps11 ( 61 ). Section title: Fixation, permeabilization, and intracellular anti-IgM staining of putative plasma cells Educational score: 3.8166894912719727 Domain: biomedical Document type: Study Language: en For detection of antibody-secreting cells, we used head kidney biopsies from non-IS fish that received either 50,000 or 250,000 S. molnari BSs. Tissue processing and staining for membrane-bound/cell surface IgM was as described above but we started with five million cells in anticipation of detecting rare cell populations. Section title: Fixation, permeabilization, and intracellular anti-IgM staining of putative plasma cells Educational score: 4.10089635848999 Domain: biomedical Document type: Study Language: en To costain for cell surface and intracellular IgM, we used the same anti-carp IgM clone WCI12 antibody specially purified from supernatant with the Mouse TCS Antibody Purification kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. The antibody was then biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s instructions with a 100-fold molar excess of biotin. The prepared antibody (WCI12-biotin) was then dialyzed against a 1000-fold volume excess of PBS. The successful biotinylation reaction was confirmed by flow cytometry and western blot using streptavidin reagents (data not shown). Section title: Fixation, permeabilization, and intracellular anti-IgM staining of putative plasma cells Educational score: 4.146445274353027 Domain: biomedical Document type: Study Language: en After cell surface staining, we washed the cells twice with Hanks’ Balanced Salt Solution (HBSS) (Thermo Fisher Scientific, Paisley, UK) supplemented with 1% fetal bovine serum. We then fixed the cells in 100 µL of IC Fixation Buffer (Thermo Fisher Scientific, Carlsbad, CA, USA) for 30 min at ambient temperature. From this point onwards, we pelleted cells by centrifugation at 800 g for 5 min. The cells were then washed twice with HBSS 1% fetal bovine serum before permeabilization for 15 min at ambient temperature with Permeabilization Buffer (Thermo Fisher Scientific, Carlsbad, CA, USA). After this fixation and permeabilization, we pelleted cells and incubated them for 30 min in Permeabilization Buffer with 5% heat-inactivated BALB/C mouse serum to saturate the existing anti-mouse IgG reagent used for membrane IgM staining. The cells were pelleted and stained in the refrigerator overnight with 300 ng of WCI12-biotin per specimen. Section title: Fixation, permeabilization, and intracellular anti-IgM staining of putative plasma cells Educational score: 3.924682378768921 Domain: biomedical Document type: Study Language: en We washed cells twice with Permeabilization Buffer. Cells were then stained with 120 µL of streptavidin PE-Cyanine7 conjugate (Thermo Fisher Scientific, Carlsbad, CA, USA) diluted 1:500 in Permeabilization Buffer for 30 min in ice. The excess streptavidin reagent was washed away twice in Permeabilization Buffer before we resuspended cells for flow cytometry analysis. Section title: Quantification and statistical analysis Educational score: 1.3177419900894165 Domain: biomedical Document type: Other Language: en All statistical details of experiments can be found in either the figure legends, or in the supplementary figures and their respective legends. Section title: Quantification and statistical analysis Educational score: 3.2380683422088623 Domain: biomedical Document type: Study Language: en Throughout this study, statistical significance was always defined by a P value of less than 0.05 or a less than 5% probability of the null hypothesis being true. We used these abbreviations to further specify the outcomes of all statistical tests we performed: ns (not significant); * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. | Review | biomedical | en | 0.999996 |
PMC11695324 | Section title: Introduction Educational score: 4.007002353668213 Domain: biomedical Document type: Review Language: en Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by difficulties in social communication, interaction, and behavioral patterns that can affect everyone, manifest typically in early childhood, and have a significant impact on both individuals and society ( 1 , 2 ). Researchers have identified approximately 1 in 68 children with some form of ASD. Despite the high global prevalence of ASD, which has increased over the past 50 years from 1:10000 live birth to 1:59 live birth, it remains incurable and, like any other childhood disorder, requires treatment and intervention. Although the precise causes are unknown, both genetic and environmental factors, including maternal factors during pregnancy, significantly influence its development ( 3 , 4 ). Section title: Introduction Educational score: 3.626279830932617 Domain: biomedical Document type: Review Language: en Many maternal factors can impact a child’s health in several ways. The uterine environment can be changed by a number of things, including polycystic ovary syndrome (PCOs), prenatal hyperandrogenism (male sex hormones), chronic anovulation, insulin resistance, metabolic syndrome, and chronic low-grade inflammation. These things may play a part in neurodevelopmental disorders like ASD, but the exact ways they cause these disorders are still not fully understood ( 5 – 7 ). Using some drugs (antidepressants, specifically selective serotonin reuptake inhibitors) during the second or third trimester of pregnancy may increase the risk of a child developing ASD ( 8 ). Section title: Introduction Educational score: 4.042227268218994 Domain: biomedical Document type: Study Language: en Several studies have indicated that mothers with gestational diabetes mellitus (GDM)-associated altered glucose metabolism and insulin resistance conditions lead to neurodevelopmental disorders such as ASD. Moreover, pregnancy-associated inflammatory processes and oxidative stress may contribute to these adverse outcomes ( 9 , 10 ). Additionally, tobacco smoke significantly impacts sperm DNA methylation and gene expression, potentially altering neuronal structure and leading to adverse birth outcomes such as low birth weight and limited growth in the fetus ( 11 ). Proper maternal nutrition during pregnancy plays a crucial role for optimal fetal development, growth, and health ( 12 ). Section title: Introduction Educational score: 3.3348772525787354 Domain: biomedical Document type: Other Language: en An autistic child prefers playing alone, avoids eye contact, has limited or repetitive patterns, and is at higher risk for injuries or abuse. This can sometimes be harmful. These deficits make it difficult for many with ASD to live independently. Therefore, family members and care providers deal with emotional, financial, and even physical stress and sexual health issues while raising a child with ASD, which appears to contribute to a general decrease in parental well-being and an increase in mental health concerns. The mother of an autistic child faces more stress, anxiety, depressive symptoms, social withdrawal, and fear compared to other mothers. Similar stages of grief, such as denial, anger, bargaining, depression, and acceptance, were experienced by others, along with a feeling of guilt as if they were responsible ( 13 – 16 ). Section title: Introduction Educational score: 2.9566476345062256 Domain: biomedical Document type: Study Language: en ASD research is scarce in developed nations like Saudi Arabia; hence, most clinical procedures are based on Western findings ( 17 ). Thus, the above-mentioned factor heightened interest in studying this topic, which is understandable. From February to May 2024, we conducted this cross-sectional study to investigate the severity level of ASD, the prenatal, maternal, and natal risk factors that might be associated with it during pregnancy, and their interrelationships among the mothers of ASD children. Section title: Study design and setting Educational score: 1.7999953031539917 Domain: biomedical Document type: Study Language: en From February 2024 to May 2024, we conducted this web-based cross-sectional study in the Society of Autism Families in SA’s large cities, Riyadh, Jeddah, and Dammam. Despite SA’s vast size, most of its services are only available in its largest cities. Section title: Participants Educational score: 4.0792341232299805 Domain: biomedical Document type: Study Language: en This cross-sectional study specifically targeted adult mothers who had received an ASD diagnosis for their children. Participants were eligible if their children were less than 18 years old, attended a pediatric outpatient clinic or an autism-specific center, and met the ASD diagnostic criteria. The American Psychiatric Association’s Diagnostic and Statistical Manual, Fifth Edition (DSM-5) provides information to help diagnose ASD. According to DSM-5 ( 16 ), a child must have persistent deficits in each of three areas of social communication and interaction, plus at least two of four types of restricted repetitive behaviors ( 15 ). Mothers with complex medical, psychological, or mental disorders were excluded as these disorders may affect recall, self-administration, and outcome accuracy. Section title: Sample size and sampling techniques Educational score: 3.838667154312134 Domain: biomedical Document type: Study Language: en We determined the sample size using the formula n = Z 2 P(1-P)/d 2 , where n stands for the sample size, Z for the desired level of confidence, P for the expected prevalence, and d for the precision or effect size. Despite a 2008 surge in local research, much remains unexplored. Saudi Arabia’s ASD prevalence is unknown. One 2002 survey found 42,500 autistic cases. Recent comprehensive reviews found that ASD prevalence in Arabian Gulf nations, including Saudi Arabia (SA), ranged from 1.4 to 29 per 10,000 ( 16 ). With a 95% confidence level and 80% power, the calculated sample size was determined to be 88 mothers of autistic children. To collect the sample, the official groups used an online Delphi panel. Section title: Data collection tool Educational score: 3.7404470443725586 Domain: biomedical Document type: Study Language: en A questionnaire was developed based on previous research ( 2 , 4 , 13 – 20 ). The questionnaire was initially drafted in English and then translated into Arabic. Two English-speaking translators performed the back translation, and they also consulted the original panel to address any issues or worries. To ensure content validity and reliability, the questionnaire underwent validation by three experts in public health, community medicine, and pediatrics. A survey was developed based on previous research ( 2 , 4 , 13 – 20 ). The questionnaire was initially drafted in English and then translated into Arabic. Two English-speaking translators performed the back translation, and they also consulted the original panel to address any issues or worries. To ensure content validity and reliability, the questionnaire underwent validation by three experts in public health, community medicine, and pediatrics. A pilot study was conducted on 10 participants who were not included in the main study to validate the questionnaire. We intentionally solicited feedback from participants, including inquiries about the clarity of the instructions, logistical, technical, and other concerns, as well as the difficulty of answering certain questions. After collecting and analyzing the pilot survey results, we addressed all the reported feedback, potentially correcting the questions or selecting the most appropriate types of queries. Cronbach’s alpha was calculated to estimate the questionnaire’s reliability, and a value of 0.78 was obtained. Section title: Data collection tool Educational score: 4.0135817527771 Domain: biomedical Document type: Study Language: en The questionnaire is composed of five main sections, following the mothers’ written informed consent [found in Supplementary Material 1 ]. The first section includes the characteristics and demographic information of both the mother and father. 2) Prenatal risk factors for autism include health and pregnancy conditions, as well as adherence to required medications. 3) Antenatal risk factors include adherence to required follow-up, maternal factors such as environmental, psychosocial, and lifestyle-related dietary habits, and physical activity. We assessed psychosocial factors using the LIVES Daily Hassles score, a tool that evaluates the impact of frequent exposure to daily life stressors like family or marital conflict, income concerns, workload, time issues, concerns about future security, and environmental or housing conditions ( 20 ). 4) Natal-related risk factors. 5) The Autism Center uses the American Psychiatric Association’s DSM-5 classification, which provides information to the mothers regarding the diagnosis and severity of autism in their children ( 16 ). Section title: The Data collection process Educational score: 2.2001099586486816 Domain: biomedical Document type: Study Language: en Data collection was carried out using a pre-tested, pre-coded, and well-structured questionnaire. We designed the questionnaire in Arabic using Google Forms and distributed it to all registered mothers of autistic children in all autism centers, leveraging popular and officially recognized social media platforms such as Facebook, Twitter, mother-registered emails, and WhatsApp groups within an autism-specific center. We sent frequent reminder messages and follow-up communications to increase the response rate until we reached the desired sample size. Section title: Statistical analysis Educational score: 3.421700954437256 Domain: biomedical Document type: Study Language: en The Jamovi software program, version 2.5.6, was used for data coding and analysis. The Shapiro-Wilk test was employed to assess the normal distribution of the data. Qualitative data was presented as frequencies and percentages, while quantitative data was displayed as mean and standard deviation (SD) if it demonstrated normal distribution or as median and interquartile range (IQR) if it did not. Section title: Statistical analysis Educational score: 3.9996397495269775 Domain: biomedical Document type: Study Language: en For examining the relationship between autism severity and continuous variables, we employed one-way ANOVA (Kruskal-Wallis). The chi-square test (χ2) and Fisher’s exact test were used to analyze the distinction between autism severity and other categorical variables, with Fisher’s exact test for sample sizes less than five and chi-square for larger sample sizes. Variables that demonstrated a significant association underwent multivariate logistic regression to assess their predictive capability for ASD severity. We considered the results statistically significant when the probability was less than 0.05. Section title: Ethical considerations Educational score: 1.2795039415359497 Domain: other Document type: Other Language: en The institutional review board (IRP) at Princess Nourah bint Abdulrahman University registered this survey under the number 24-0363 and gave its approval on February 19, 2024. Before participating in the Google forum, we requested that all invited participants complete and check an informed consent box. Participation in the survey was voluntary and related to the autism center. Checking the consent box and completing the voluntary survey were considered evidence of providing valid, informed consent. The IRB application for this study, which received approval, described this consent process. Section title: Regarding the frequency or severity of autism Educational score: 2.493607759475708 Domain: biomedical Document type: Study Language: en Regarding the frequency or severity of autism, a survey of 168 mothers with children with ASD revealed that 15.6% of these children had mild autism, 31.9% had moderate autism, 43.8% had autism spectrum disorder, and 8.8% had severe autism. Section title: Demographics and prenatal history, and their correlation with the severity grades of ASD Educational score: 3.6756770610809326 Domain: biomedical Document type: Study Language: en The median maternal age was 30 years, and the median paternal age was 35 years. Most participants resided in urban areas, 138 (86.3%), with slightly higher proportions in the mild and severe autism categories. In descending order regarding prenatal history or risk factors, 23 (14.4%) had ovarian cysts before pregnancy, 11 (6.9%) used assisted reproductive techniques (IVF-tubal pregnancy), 4 (2.5%) had type 1 diabetes, and 2 (1.3%) had type 2 diabetes before pregnancy. There was no statistically significant difference between the severity grades of ASD, demographics, and prenatal history ( Table 1 ). Section title: The relationship between the severity grades of ASD and the antenatal maternal and paternal exposure history or maternal antenatal conditions (medical and environmental factors) Educational score: 2.824014186859131 Domain: biomedical Document type: Study Language: en Most autistic children had a history of one or both maternal and/or paternal antenatal exposures, as follows, in descending order: 77.7% had soft drink consumption, 30.6% smoked, 20.7% had chronic physical organic diseases (e.g., high blood pressure, asthma), 16.9% had psychological or neurological disease, and 13.3% had a history of diet product consumption (aspartame). Section title: The relationship between the severity grades of ASD and the antenatal maternal and paternal exposure history or maternal antenatal conditions (medical and environmental factors) Educational score: 3.895885705947876 Domain: biomedical Document type: Study Language: en Regarding maternal antenatal conditions, the most common events, in descending order, were: 33.7% history of recurrent infection; 25.7% anemia during pregnancy; and 26 (16.3%) threatened abortion or bleeding during pregnancy. During pregnancy, there is a risk of exposure to air pollution, which includes elevated carbon levels, 22 (13.8%) cases of gestational diabetes, 14 (10.7%) individuals taking medications for chronic conditions such as insulin, thyroxine, corticosteroids, and hydroxychloroquine, and 8 (5%) individuals receiving the COVID-19 vaccine and antibiotics such as amoxicillin, cotrimoxazole, and penicillin. Section title: The relationship between the severity grades of ASD and the antenatal maternal and paternal exposure history or maternal antenatal conditions (medical and environmental factors) Educational score: 3.9711363315582275 Domain: biomedical Document type: Study Language: en The study found no statistically significant difference between the severity grades of ASD, antenatal maternal and paternal exposure history, or maternal antenatal conditions ( p > 0.05), except for gestational diabetes during pregnancy ( p = 0.047). The results showed that 4 (28.6%) of 14 cases had severe autism, 8 (15.7%) of 50 cases had moderate autism, and 0 (0%) cases had mild autism. As illustrated in detail in Table 2 . Section title: Maternal lifestyle factors (including dietary and physical activity during pregnancy) and their relationship with the severity grades of ASD Educational score: 2.7146942615509033 Domain: biomedical Document type: Study Language: en Although approximately 45% of mothers didn’t remember their average daily intake, the majority reported less than the daily recommended diet of milk and dairy products. 67, 41.9%/94: vegetables 66, 41.3%/91: fruit 65, 65, 40.6%/93: water consumption 115, 71.9%): grains and bread 40, 25%/64; meat and legumes 43, 26.9%/85. The level of physical activity was 102 (63.7%). Routine household activities. The study found no significant association between the severity of ASD and the nutrition and lifestyle factors during pregnancy. As illustrated in detail in Table 3 . Section title: LIVES Daily Hassles Factors and Scale (psycho-social factors) during pregnancy and their relationship with the severity grades of ASD Educational score: 3.7912614345550537 Domain: biomedical Document type: Study Language: en In descending order, the majority reported that daily hassles, including household chores, spouse conflict, time pressure, financial concerns, internal fears, and work-related factors, had a negative impact on their health. The study found that there was no significant correlation between the severity of autism spectrum disorder (ASD) and the LIVES Daily Hassles Factors and Scale during pregnancy. However, the impact of household chores on health during pregnancy was significantly (P = 0.02) higher among individuals with severe autism (around 14.3%), compared to those with mild autism (12%) or moderate autism (5.9%). As illustrated in detail in Table 4 . Section title: The natal history, ASD diagnosis history, and correlation with ASD severity grades Educational score: 3.5863099098205566 Domain: biomedical Document type: Study Language: en In terms of natal history and risk factors, there were 104 (65%) autistic children born in the government hospital. Out of these, 97 (60.6%) were born vaginally, 21.2% had an abnormal birth weight, 50 (31.3%) required admission to the neonatal intensive care unit, 23 (14.4%) had a history of oxygen desaturation, 11.9% had an abnormal birth length, and 16 (10%) had a history of any or recurrent infections. Only 88 (55%) cases with a history of autism received a diagnosis based on symptoms, with 82 (51.2%) families serving as the primary caregivers. Section title: The natal history, ASD diagnosis history, and correlation with ASD severity grades Educational score: 4.061793327331543 Domain: biomedical Document type: Study Language: en The study found no significant correlation between the severity of ASD and the natal history and ASD diagnosis. The study did, however, find a strong link (P = 0.021) between a child’s length at birth—which was higher at 3 (21.4%) in cases of severe autism compared to other grades less than 10%—and their oxygen desaturation—which was higher at 6 (42.9%) in cases of severe autism compared to other grades less than 14.3% (p = 0.017). In addition, the current median age of the child (years) is significantly ( p = 0.035) higher in severe autism cases at 14 (12.25–18) compared to the median mild autism 7 (5.5–13), compared to the median moderate as well as autism spectrum. As illustrated in detail in Table 5 . Section title: In terms of antenatal history Educational score: 4.038538932800293 Domain: biomedical Document type: Study Language: en In terms of antenatal history, gestational diabetes during pregnancy is a strong predictor of severe autism. The aOR for the severity of ASD among women with gestational diabetes during pregnancy relative to those without gestational diabetes during pregnancy is 4.553 (95% CI: [1.518, 14.25], P = 0.008) ( Table 6 ). This suggests that women with gestational diabetes during pregnancy are approximately 4.5 times more likely to have children with severe ASD compared to those without gestational diabetes during pregnancy. Section title: Regarding natal history a child’s oxygen desaturation Educational score: 4.021756649017334 Domain: biomedical Document type: Study Language: en Regarding natal history A child’s oxygen desaturation is a significant predictor of severe autism. The aOR for the severity of ASD among children with oxygen desaturation relative to those without oxygen desaturation is 4.142 (95% CI: [1.437, 12.45], P = 0.01). This suggests that children with oxygen desaturation are approximately 4.1 times more likely to develop severe ASD than those without. Section title: In terms of autism history Educational score: 4.000019550323486 Domain: biomedical Document type: Study Language: en In terms of autism history, a child’s current age in years is a significant predictor of severe autism. The aOR for the severity of ASD associated with each additional year of the child’s age is 1.071 (95% CI: [1.002, 1.15], P = 0.045). This indicates that for every one-year increase in the child’s age, the odds of being in a higher category of ASD severity relative to a lower category increase by approximately 7.1%. Section title: Discussion Educational score: 2.4401490688323975 Domain: biomedical Document type: Study Language: en In SA, there is a significant gap between the demands of those diagnosed with ASD and the currently offered services ( 17 ). Early intervention focuses on language, play skills, social involvement, activities of daily living (ADLs), and disruptive behaviors. Genetic and epigenetic factors may contribute to ASD’s multifactorial origin ( 21 ). As a result, this local study from SA is critical to highlighting prenatal, antenatal, and natal-related factors, or associated ASD. Section title: The frequency or severity of autism Educational score: 3.14524245262146 Domain: biomedical Document type: Study Language: en In this study, the majority of the recruited 168 mothers with Autistic children reported having autism spectrum disorders (43.8%). Moderate autism accounts for 31.9%, mild autism for 15.6%, and severe autism (8.8%). According to the global classification of ASD severity, most mothers reported having ASD, a broader category that encompasses all levels of severity, including moderate, mild, and severe autism. Remember, diagnostic practices are crucial. Section title: The frequency or severity of autism Educational score: 3.227870225906372 Domain: biomedical Document type: Review Language: en Rates of occurrence may be different depending on diagnostic criteria, cultural contexts, and research methods; without detailed sub-categorization, this isn’t standard practice ( 22 ). For example, a narrative review of UK clinical practice guidelines for ASD diagnosis shows that diagnostic rates vary depending on social and contextual factors ( 23 ). Furthermore, the level of maternal awareness and understanding were relatively low, with only 56.2% being able to identify and comprehend their child’s subcategorical classification. Section title: Parental age and ASD Educational score: 3.890126943588257 Domain: biomedical Document type: Study Language: en Although there was a non-significant association between parental age and ASD severity, those with severe autism had a relatively older median (IQR) parent age of 39 years (30–45.8), while other groups were 35 years. This may be due to the relatively small number of severe autism cases (8.8%). Previous research links older parental age to higher ASD risk in children, potentially due to genetic mutations in older gametes and exposure to various environmental factors over a longer period, some of which could impact the health and development of their offspring ( 5 – 24 ). Section title: Polycystic ovarian syndrome Educational score: 4.063864707946777 Domain: biomedical Document type: Study Language: en We found that 23 mothers (13.7%) of children with ASD had history of PCOS, with an average of 5% to 20% of PCOS among women of reproductive age and no significant correlation with the severity grades. In agreement with a large-scale study from Sweden, PCOS has identified a potential increased risk of birthing children with ASD. Although PCOs underlying pathogenic mechanisms have not been completely understood, prenatal hyperandrogenism (male sex hormones; chronic anovulation, insulin resistance, metabolic syndrome, chronic low-grade inflammation)—all these factors may affect the intrauterine environment and may play a role in neurodevelopmental disorders, including ASD ( 6 – 25 ). Section title: Soft drink consumption Educational score: 2.490086793899536 Domain: biomedical Document type: Study Language: en Out of 168 mothers (8.3%), 81 (48.2%) reported that both fathers and mothers drank soft drinks, and 19% had a history of diet product consumption (aspartame). The FDA approved aspartame as a tabletop sweetener in 1981. By 1983, the FDA had reported many adverse events, e.g., depression, anxiety, headache complaints, and other neurological problems, including irritability, mood disorders, cognitive problems, and seizures ( 26 , 27 ). Section title: Soft drink consumption Educational score: 4.149744510650635 Domain: biomedical Document type: Study Language: en Moreover Aspartame-fed animals displayed cognitive problems, anxiety-related behaviors, and other noticeable neurophysiologic effects. This has led to the discovery of long-term health problems in humans and their offspring, as the availability of glutamine sulfhydryl (GSH) has greatly decreased. This is significant because GSH protects the developing brain by scavenging toxins, preventing oxidative stress, and supporting methylation. There are more free radicals, oxidative stress, lipid peroxidation, inflammation, mitochondrial dysfunction, excitotoxicity, neuronal apoptosis, serotonin, noradrenaline, and dopamine in the brain when aspartame and its byproducts are present ( 27 – 29 ). Section title: Maternal smoking history Educational score: 3.9788224697113037 Domain: biomedical Document type: Study Language: en Maternal smoking history: 35.1% out of 168 mothers reported that fathers were smoking. The adjusted odds ratio (aOR) indicates the strength of this association, as well as a higher likelihood of having offspring with ASD ( 11 ). In agreement with other systematic reviews, tobacco smoke is the poisonous substance that most likely harms brain development. Nicotine affects nicotinic acetylcholine receptors in the offspring later in life, which mediate neural structural changes that can lead to adverse birth outcomes such as fetal growth restriction and low birth weight. Moreover, it significantly alters sperm DNA methylation and gene expression ( 11 ). Section title: Medication use during pregnancy Educational score: 3.323017120361328 Domain: biomedical Document type: Study Language: en Medication use during pregnancy We did not identify a notable association between medication use during pregnancy and ASD; however, other studies have suggested that the use of certain substances might be a risk factor for ASD. For instance, a study suggested that the use of antidepressants, specifically selective serotonin reuptake inhibitors, during the second and/or third trimesters increases the risk of ASD ( 8 ). Section title: Medication use during pregnancy Educational score: 3.943777322769165 Domain: biomedical Document type: Study Language: en Among ASD mothers, only 9 (5.4%) consume prenatal vitamins and supplements (e.g., folic acid, calcium, iron, vitamin B12, and vitamin D). This indicates that consuming prenatal vitamins and supplements reduces the risk of ASD, and certain nutrients may have neuroprotective effects throughout development. This aligns with a systematic review that found that maternal prenatal vitamins, folic acid, and vitamin D reduce the risk of autism in offspring. Higher maternal intakes of folic acid, omega-6 fatty acids, and vitamin D during pregnancy reduced child autism risk, while women deficient in omega-3 and polyunsaturated fatty acids raised it ( 30 , 31 ). Section title: Infections and anemia during pregnancy Educational score: 4.013270378112793 Domain: biomedical Document type: Study Language: en During pregnancy, 29 out of 168 mothers (17.3%) reported having recurrent infections. The nested case-control study found no overall significant association between any maternal infection during pregnancy and infections diagnosed during hospital admission, particularly bacterial infections ( 32 ). The lower level of immunity, along with the associated anemia, may pose a potential risk for ASD in 30 out of 168 mothers (17.9%). In alignment with other research, mothers diagnosed with anemia within the first 30 weeks of pregnancy had a higher prevalence of ASD, ADHD, and intellectual disabilities ( 33 , 34 ). Section title: Nutrition during pregnancy Educational score: 3.6115381717681885 Domain: biomedical Document type: Study Language: en Although approximately 45% of mothers didn’t remember their average daily intake, the majority reported less than the daily recommended diet of milk and dairy products. 67, 78.7%/94: vegetables 66, 72.5%/91: fruit. 65, 69.9%/93: water, followed by grains and bread at 40, 60%/64, and meat and legumes at 45, 51.7%/87. This recall may indicate that the mothers did not follow an appropriate diet, which could potentially influence the risk of ASD. This pattern of dietary consumption is consistent with another two studies in SA ( 35 , 36 ). This result aligns with the findings of another two United States cohorts, which suggested a positive association between the Western diet and autism-related traits ( 37 ). Section title: Nutrition during pregnancy Educational score: 4.083251953125 Domain: biomedical Document type: Review Language: en Here are some key points highlighting the importance of nutrition during pregnancy: 1. Fetal Development: Adequate intake of nutrients such as folic acid, iron, calcium, and omega-3 fatty acids supports the development of the baby’s brain, bones, and overall body structure. 2. Birth Defect Prevention: Proper nutrition can help prevent birth defects. For instance, folic acid is known to reduce the risk of neural tube defects. 3. Healthy Birth Weight: A balanced diet helps to achieve a healthy birth weight, reducing the risk of complications during delivery and ensuring the baby has a favorable start in life. 4. Immune System Support: Nutrients like vitamins A, C, and E, along with zinc, support the development of the baby’s immune system, helping to protect against infections. 5. Long-term Health: Maternal nutrition can have long-term effects on the child’s health, influencing their risk of developing chronic conditions such as obesity, diabetes, and cardiovascular diseases later in life. 6. Cognitive Development: Essential fatty acids, particularly DHA, are critical for the development of the baby’s brain and eyes, potentially impacting cognitive function and vision ( 12 ). Section title: The study focuses on the Daily Hassles Factors, their scale during pregnancy, and their relationship with the severity grades of ASD Educational score: 4.078469753265381 Domain: biomedical Document type: Study Language: en In descending order, the majority reported that there was a negative impact on the health of the daily hassles: 78.5% of household chores, 72.6% of spouse conflict, 67.8% of time pressure, 53.6% of financial concerns, 51.8% of internal fears, and 27.9% of concerns about the future, as well as work-related factors. There was no significant correlation between the severity of ASD and the LIVES Daily Hassles Factors and Scale during pregnancy, according to the study. However, the impact of household chores on health during pregnancy was significantly ( P = 0.02) higher among individuals with severe autism (around 14.3%), compared to those with mild autism (12%) or moderate autism (5.9%). Household chores and pregnancy stress: In our study, we identified a notable association between the stress from household chores during pregnancy and ASD development. This underscores the potential impact of prenatal stress on fetal development, possibly through hormonal changes or other stress responses. Our findings call for more support for expectant mothers to reduce stress and physical strain, which may lower ASD risk ( 38 , 39 ). Section title: The history of ASD diagnosis and its relationship to ASD severity grades Educational score: 4.0532097816467285 Domain: biomedical Document type: Study Language: en Two major domains of ASD diagnosis are social and communication interaction and restricted or repetitive conduct. In this study, only 88 (55%) cases received a diagnosis based on their symptoms, while 82 (51.3%) families handled the diagnosis. SA-related methodological issues, poor diagnostic competence, and lower parent awareness of ASD can all contribute to this gap, limiting the likelihood of recognizing symptoms and seeking therapy. Parents of autistic children face a lack of government and institutional support, as well as a lack of resources for autistic children, which may affect other siblings’ quality of life, their social and economic lives, family ties. In Saudi Arabia, 88.5% of parents of children with autism experience discomfort; out of 61 families with ASD, 37% felt ashamed for having autistic children, and 64% were concerned about others’ treatment. According to SA Khan et al.’s 2020 cross-sectional study, only 31% of children on the autistic spectrum could attend neighboring autism clinics, and 72% had no access to private ASD schools ( 40 – 42 ). Section title: The history of ASD diagnosis and its relationship to ASD severity grades Educational score: 3.971081018447876 Domain: biomedical Document type: Study Language: en Radiological and laboratory tests diagnosed only 29 (18.1%) ASD cases, primarily diagnosing 16 (55%) cases of autism spectrum disorder. While SA recommends genetic testing for all ASD patients, chromosomal microarray analysis (CMA) is more effective when the cause of developmental delay is unknown, as 20-30% of individuals on the autism spectrum exhibit significant genetic variation and new mutations. Moreover, consanguinity in Saudi culture—57.7% of marriages—was considered in these recommendations ( 43 , 44 ). Section title: Gestational diabetes mellitus Educational score: 4.205443382263184 Domain: biomedical Document type: Study Language: en In this study, 22 out of 168 mothers (13.1%) reported having GDM, and there was a statistically significant association ( p -value of 0.047) between GDM and the severity of ASD. Numerous mechanisms explain this association and GDM’s role. 1) GDM influences placental function, which in turn alters the delivery of nutrients and oxygen to the fetus, leading to hypoxia. Hypoxia (the developing brain doesn’t receive enough oxygen) can impair neurogenesis and lead to neuronal cell death, thereby contributing to neurodevelopmental impairments and potentially increasing the severity of ASD. Hypoxia can lead to high blood sugar levels, which can cause oxidative stress and inflammation in the developing fetal brain. 2) High blood sugar increases the production of reactive oxygen species, leading to oxidative stress, which can harm cellular structures such as lipids, proteins, and DNA, potentially disrupting normal brain development. 3) High blood sugar-induced inflammation can trigger the release of pro-inflammatory cytokines, potentially disrupting neuronal development and synaptic plasticity. These inflammatory processes can worsen the severity of ASD by further impairing neural connectivity and function ( 10 , 45 , 46 ). Moreover, this underscores the importance of closely monitoring and managing gestational diabetes during pregnancy. Section title: Oxygen desaturation and ASD Educational score: 4.046265602111816 Domain: biomedical Document type: Study Language: en Children exposed to oxygen desaturation at birth are more likely to develop severe ASD, according to a p -value of 0.017. This aligns with previous studies ( 45 – 49 ). This finding highlights the importance of early interventions for children with perinatal complications to potentially reduce ASD severity ( 47 , 50 ). However, researchers are still exploring the exact pathological mechanisms that link oxygen desaturation to ASD. Hypoxia can cause inflammation, oxidative stress, and brain development disruptions, which may contribute to the development of ASD ( 4 , 51 ). Section title: Child’s age and ASD severity level Educational score: 3.065631628036499 Domain: biomedical Document type: Study Language: en A 7.1% increase in the likelihood of classifying a child’s ASD as more severe with each year. This suggests that the challenges associated with ASD may become more pronounced as children age. However, it’s important to note that ASD symptoms can change over time, with some children showing a decrease in severity. This variability indicates that ASD is a dynamic condition, and interventions should be adaptable to each child’s changing needs ( 48 ). Section title: Strengths and limitations Educational score: 3.9771006107330322 Domain: biomedical Document type: Study Language: en This study has many strengths, including the relatively large sample size, the use of a validated tool, the collection of paternal and maternal factors, and the comprehensiveness of prenatal, antenatal, and natal medical, environmental, dietary, physical, and psychosocial factors. Furthermore, we investigated the effect of frequent exposure to seven daily life stressors on health, which include family or marital conflict, income concerns, workload, time issues, concerns about future security, and environmental or housing conditions. Section title: Strengths and limitations Educational score: 3.7409539222717285 Domain: biomedical Document type: Study Language: en Although exploring the risk factors for autism with unknown causes is critical, the study has many limitations. For example, the study’s cross-sectional design introduces recall biases, as approximately 50% of mothers were unable to recall their dietary habits. Furthermore, the subjective report highlights the health effects of frequent exposure to seven daily life stressors during pregnancy. The web-based survey employs Delphi sampling techniques and excludes mothers with complex medical, psychological, or mental disorders, potentially influencing the results’ generalizability. Section title: Conclusion Educational score: 2.7331383228302 Domain: biomedical Document type: Study Language: en This study highlighted the maternal risk factors associated with autism spectrum disorders (ASD) in children. ASD is a diverse group of conditions characterized by some degree of difficulty with social interaction and communication. Section title: Conclusion Educational score: 4.017826080322266 Domain: biomedical Document type: Study Language: en Our study has shown a significant connection between gestational diabetes and autistic children, acknowledging the importance of interventions, screening tests, and lifestyle modifications during pregnancy. We also found a link between the severity of ASD and oxygen desaturation at birth. This highlights the importance of intervention in such cases to improve the child’s quality of life. Moreover, the mother’s stress and physical activity during household chores showed increased development of ASD in children, but a wider study is necessary to confirm the exact mechanism. Finally, the child’s age. The odds of having a more severe ASD increase with age. Section title: Recommendation Educational score: 2.284987211227417 Domain: biomedical Document type: Study Language: en After conducting this study, we concluded that understanding the relationship between the discussed factors and ASD development in children enables us to more effectively direct support services to meet the needs of these mothers and their children, enhancing their overall well-being. | Review | biomedical | en | 0.999998 |
PMC11695325 | Section title: Introduction Educational score: 3.813831090927124 Domain: biomedical Document type: Review Language: en Hepatitis B is a viral infection that primarily affects the liver, leading to both acute and chronic diseases ( 1 ). The World Health Organization (WHO) has recognized the urgency of addressing this issue, as outlined in its Global Hepatitis Report, which highlights the need for strategic plans to eliminate viral hepatitis as a public health threat by 2030, despite these efforts, an estimated 250–296 million people worldwide are living with chronic Hepatitis B virus (HBV) infections, making it a leading cause of cirrhosis and liver cancer ( 2 , 3 ). Section title: Introduction Educational score: 3.739851474761963 Domain: biomedical Document type: Study Language: en Each year, approximately 1.5 million new infections are diagnosed, with HBV responsible for an estimated 1.1 million deaths in 2022, primarily due to complications like cirrhosis and hepatocellular carcinoma. The disease is often referred to as a “silent epidemic” due to its asymptomatic nature in many individuals ( 4 , 5 ). In Africa, around 82.3 million people are affected by chronic HBV, with Sub-Saharan Africa facing one of the highest burdens of the disease—over 60 million individuals are living with HBV ( 5 ). Section title: Introduction Educational score: 3.33864688873291 Domain: biomedical Document type: Study Language: en In Ethiopia, studies indicate that the prevalence of Hepatitis B surface antigen (HBsAg) ranges from 5.4% to 12.7%. Alarmingly, around 95% of those with chronic HBV are unaware of their infection, equating to over 5 million people in the general population ( 6 , 7 ). Despite having a comprehensive national plan to investigate and control viral hepatitis, Ethiopia remains categorized as a region with hyper-endemic HBV infections ( 8 ). Section title: Introduction Educational score: 2.0560967922210693 Domain: biomedical Document type: Other Language: en Awareness of HBV is notably low in the United States, with only 32% of the population informed about the disease, this gap underscores the need for enhanced education to prevent the spread of HBV ( 9 ). Section title: Introduction Educational score: 2.6901285648345947 Domain: biomedical Document type: Study Language: en Studies indicate that healthcare workers often lack adequate training in infection control measures, increasing transmission risks. A multi-institutional study across African countries revealed low awareness and vaccination rates among healthcare workers, highlighting significant gaps in understanding HBV prevention ( 2 , 10 ). Section title: Introduction Educational score: 2.827543020248413 Domain: biomedical Document type: Study Language: en In Ethiopia, the pooled prevalence of good practices related to the HBV stands at 41.5% (95% CI: 30.8%–51.6%) ( 11 ). Factors influencing awareness include age, education level, and attitude. Conversely, the levels of practice are affected by sex, educational status, residence, occupational status, patient condition, and vaccination status ( 12 – 17 ). Section title: Introduction Educational score: 2.3891706466674805 Domain: biomedical Document type: Study Language: en In Zambia, research on hospital practices has revealed that informal caregivers demonstrate poor practices regarding HBV prevention. This population is at heightened risk of exposure to HBV due to their close interactions with infected individuals and often lacks access to proper protective measures or adequate information about prevention strategies ( 18 ). Section title: Introduction Educational score: 2.3801474571228027 Domain: biomedical Document type: Study Language: en Despite the critical role of informal caregivers, who provide care without formal training, there is a lack of evidence of their awareness and practices related to hepatitis b infection prevention. Therefore, this study aims to asses awareness and infection prevention practices of Hepatitis B Virus among informal caregivers in public hospitals in Addis Ababa, Ethiopia, in 2024. Section title: Study area and period Educational score: 1.9699831008911133 Domain: biomedical Document type: Study Language: en The study was conducted in Addis Ababa, the capital city of Ethiopia, with an estimated population size of 5,703,628 in 2024. Located at the foot of Mount Entoto, at an altitude of 2,355 m above sea level. According to the data obtained from the Addis Ababa City Administration Health Bureau ( 19 ). There are 13 public hospitals in Addis Ababa, which provide different services to the public. Seven hospitals (Tikur Anbessa Specialized Hospital, Zewditu Memorial Hospital, Ras Desta Damtaw Memorial Hospital, Yekatit 12 Hospital, St Paul Hospital, Menilik Referral Hospital, and St Peter Hospital) that give hepatitis B service were randomly selected. The study was conducted from May 15 to July 15, 2024. Section title: Study design Educational score: 2.053974151611328 Domain: biomedical Document type: Study Language: en An institution-based prospective cross-sectional was employed. Section title: Source of population Educational score: 1.674875020980835 Domain: biomedical Document type: Other Language: en All adult caregivers of HBV infected patients who attended public hospitals in Addis Ababa, where the source population. Section title: Study population Educational score: 1.7346522808074951 Domain: biomedical Document type: Study Language: en All randomly selected adult informal caregivers of HBV infected patients who attend a selected public hospital in Addis Ababa. Section title: Inclusion criteria Educational score: 1.6313765048980713 Domain: biomedical Document type: Other Language: en All informal caregivers of HBV infected patients who are greater than 18 years old. Section title: Exclusion criteria Educational score: 1.6619442701339722 Domain: biomedical Document type: Other Language: en Informal caregivers of newly HBV-diagnosed patients. Section title: Sample size determination and sampling technique Educational score: 3.5708611011505127 Domain: biomedical Document type: Study Language: en The sample size of the study was determined using the single population proportion formula. Taking into account a 95% confidence interval (CI) and a 5% margin of error (d), along with accounting for a prevalence rate of 50% for the practice of infection prevention among informal caregivers in Ethiopia, due to no previous studies, and considering a 10% non-response rate, the final sample size was 422. Section title: Sampling procedure Educational score: 3.752004861831665 Domain: biomedical Document type: Study Language: en A random sampling technique was employed to select seven hospitals. The sample size was proportionally allocated to each hospital based on the total number of hepatitis B-infected patients visited in the last six months before data collection. The total number of HBV-infected patients ( N ) was determined (1,028). A proportional allocation factor (Nh) was calculated ( n / N ). This factor (0.411) was multiplied by the number of HBV-infected patients in each hospital (ni) to determine the sample size allocated to each hospital (nh). Section title: Sampling procedure Educational score: 2.7042434215545654 Domain: biomedical Document type: Study Language: en To select the required sample, in each hospital, systematic sampling techniques were used. First, the average six-month number of HBV-infected patients visiting the selected hospital has been determined which is 1,028. Every k interval is calculated i.e., k = 1,028/422 = 3, so that every third informal caregiver is included until the required sample size is achieved. The first informal caregiver was taken using the lottery method. Section title: Operational definition Educational score: 2.2334349155426025 Domain: biomedical Document type: Other Language: en Informal caregivers are individuals who provide unpaid, ongoing assistance with activities of daily living (ADLs), such as toileting, feeding, bathing, walking, and dressing, as well as instrumental activities of daily living (IADLs) ( 20 ). Section title: Operational definition Educational score: 2.1265265941619873 Domain: biomedical Document type: Study Language: en Good Awareness were those participants who scored at or above the median value on the awareness question, while, participants who scored below the median value on the awareness question were categorized as poor awareness ( 17 ). Section title: Operational definition Educational score: 1.9491136074066162 Domain: biomedical Document type: Study Language: en Respondents scoring at or above the median on attitude assessment questions were classified as having a positive attitude, while those below the median were labeled as having a negative attitude. Similarly, participants scoring at least the median on practice assessment questions were categorized as having good practice, and those scoring below were classified as having poor practice ( 17 , 21 ). Section title: Independence level in daily living activity Educational score: 3.686647653579712 Domain: biomedical Document type: Other Language: en The index ranks adequacy of performance in six areas of self-care: bathing, dressing, toileting, transferring, continence, and feeding. A score of 6 indicates a full or satisfactory function for all 6 self-care domains, and a score less than 6 indicates the presence of self-care deficit (3–5 indicates moderate impairment, and 2 or less indicates severe functional impairment) ( 22 ). Section title: Data collection instruments and procedures Educational score: 2.5290896892547607 Domain: biomedical Document type: Study Language: en The data collection tools were developed based on a review of relevant literature ( 12 – 17 ). The tools included sections on socio-demographic characteristics of caregivers, patient-related characteristics, awareness-related items, practice-related items, and attitude-related items. The questionnaire was originally prepared in English and then translated into Amharic languages. A language expert performed a back-translation into English to ensure consistency. Data were collected through face-to-face interviews conducted by a team of six bachelor-level nurses, supervised by four experienced supervisors—two of whom held master's degrees. Before the interviews, respondents received a brief orientation about the study's purpose. Section title: Data collection instruments and procedures Educational score: 2.1077001094818115 Domain: biomedical Document type: Study Language: en Data were collected by a team of six bachelor-level nurses under the supervision of four supervisors—two with master's degrees through interviews. The internal consistency of the items related to awareness, attitudes, and practices was assessed, yielding Cronbach alpha values of 0.73, 0.77, and 0.81, respectively. Section title: Data quality control Educational score: 2.44480299949646 Domain: biomedical Document type: Study Language: en To ensure data quality and uphold ethical standards, a comprehensive two-day training was conducted for data collectors and supervisors. This training encompassed the study's objectives, and ethical considerations including informed consent, confidentiality, the right to withdraw from the study, and effective data collection methods. The data collection tools were translated into Amharic, and a pretest was administered to 21 participants, representing 5% of the total sample size, at Hawasa Specialized Hospital. Based on the feedback from the pretest, necessary adjustments were made to the tools to enhance clarity and relevance. During the actual data collection, supervisors closely monitored the data collectors. They were instructed to ensure that each questionnaire was complete and that participants understood their rights, including the importance of their voluntary participation and the confidentiality of their responses. Section title: Data analysis and processing Educational score: 4.033308029174805 Domain: biomedical Document type: Study Language: en Data were entered and cleaned using EpiData version 4.6, and statistical analysis was performed using SPSS version 26. Descriptive statistics were employed to summarize the participants’ characteristics, with the results presented in text, tables, and figures. A binary logistic regression model was used to explore the association between each independent variable and the outcome variable. Independent variables with a p -value of less than 0.25 in the bivariate analysis were included in the multivariable analysis. Multi-collinearity was assessed using the variance inflation factor and standard error, while the model's fitness was evaluated using the Hosmer-Lemeshow goodness-of-fit test. Adjusted odds ratios (AOR) with 95% confidence intervals (CI) were reported, and a p -value of less than 0.05 was considered statistically significant. Section title: Socio-demographic characteristics Educational score: 2.502713441848755 Domain: biomedical Document type: Study Language: en The study included a total of 414 informal caregivers, achieving a response rate of 98.1%. The mean age of the caregivers was 34.31 years (±12). Among them, 32.9% were under 25 years of age, followed by 28.3% who were between 25 and 34 years old. The majority of respondents were single (47.6%), and 63% were female. Additionally, more than half of the caregivers (51%) had completed primary education. A significant proportion (38.2%) were engaged in trade or commerce, and approximately three-quarters reported a monthly income ranging from 1,500 to 5,000 ETB. Nearly all respondents (89.6%) resided in urban areas ( Table 1 ). Section title: Patient-related characteristics Educational score: 3.834926128387451 Domain: biomedical Document type: Study Language: en The mean age of the patients in this study was 54.5 years (±19.7 years). Nearly half of the patients (49.5%) were over 44 years old, and 54.1% were female. A significant portion (58.9%) of the patients had health insurance, and 61.8% were found to have severe impairments in daily living activities. Nearly half (45.7%) were treated in the outpatient department, while 40.6% received care in an inpatient setting. Acute hepatitis B virus infection was observed in 44.9% of the patients, and a majority (67.1%) had comorbidities, with HIV (48.2%) being the most common, followed by diabetes mellitus (32.4%) ( Table 2 ). Section title: The measure of variation and central tendency for awareness, attitude, and practice of respondents towards hepatitis B infection prevention Educational score: 2.606403112411499 Domain: biomedical Document type: Study Language: en The study revealed that the mean score for practice assessment questions was 2.068, while the mean score for awareness was 4.613. Additionally, the median score for attitudes was 4.000. The standard deviations for practice, attitude, and awareness were found to be 0.66, 1.995, and 1.77, respectively ( Table 3 ). Section title: Awareness of caregivers toward hepatitis B infection prevention Educational score: 2.6876676082611084 Domain: biomedical Document type: Study Language: en In this study, 103 caregivers (24.9%; 95% CI: 20.7%–29.1%) demonstrated good awareness of HBV infection prevention . Section title: The attitude of caregivers towards hepatitis b virus infection prevention Educational score: 2.5559985637664795 Domain: biomedical Document type: Study Language: en The study revealed that 217 caregivers (52.4%; 95% CI: 47.6%–57.3%) exhibited a positive attitude towards HBV infection prevention, while the remaining 47.6% demonstrated a negative attitude . Section title: Hepatitis B virus infection prevention practice Educational score: 2.7895703315734863 Domain: biomedical Document type: Study Language: en The study found that 48 (11.6% (95% CI: 8.5, 14.7%) of caregivers demonstrated good practice in HBV infection prevention, and the remaining 366 (88.4%) were found to have poor practice. Section title: Factors associated with awareness of caregivers toward hepatitis B infection prevention Educational score: 3.9869675636291504 Domain: biomedical Document type: Study Language: en In the bivariable analysis, factors such as caregivers' education, marital status, attitude and practice towards HBV infection prevention, level of physical activity, disease type, and comorbidities of their patients were statistically significant with a P -value less than 0.25. However, in the multivariable analysis, caregivers' attitudes and practices towards HBV infection prevention, as well as the level of physical activity of their patients, remained statistically significant at P < 0.05. Section title: Factors associated with awareness of caregivers toward hepatitis B infection prevention Educational score: 4.04236364364624 Domain: biomedical Document type: Study Language: en The odds of having a good awareness of HBV infection prevention were nearly twice as high [AOR 1.9; 95% CI (1.03–3.5)] among informal caregivers whose patients were fully functional compared to those with severe impairment. Caregivers with a positive attitude towards infection prevention were about three times more likely to have good awareness [AOR 2.54; 95% CI (1.4–4.72)] compared to those with a negative attitude. Moreover, informal caregivers who demonstrated good practices regarding HBV infection prevention were four times more likely to have good awareness [AOR 4.2; 95% CI (1.9–9.01)] compared to those with poor practices ( Table 4 ). Section title: Factors associated with the practice of caregivers toward hepatitis B infection prevention Educational score: 3.9588844776153564 Domain: biomedical Document type: Study Language: en In the bivariable analysis, factors such as caregivers' education, level of awareness, attitude towards HBV infection prevention, hospital setting, and the disease type of their patients were statistically significant with a P -value less than 0.25. However, in the multivariable analysis, caregivers’ educational status, awareness and attitudes towards HBV infection prevention, and the disease type of their patients remained statistically significant at P < 0.05. Section title: Factors associated with the practice of caregivers toward hepatitis B infection prevention Educational score: 4.047451496124268 Domain: biomedical Document type: Study Language: en The odds of having good practices in HBV infection prevention were nearly five times higher among informal caregivers with secondary education or above compared to those with no formal education [AOR 4.84; 95% CI (1.22–11.1)], [AOR 5.3; 95% CI (1.53–13.3)], respectively. The odds of good practice were about four times higher [AOR 3.6; 95% CI (1.8–8.9)] among informal caregivers whose patients had acute disease compared to those with chronic disease. Caregivers with a positive attitude towards infection prevention were more likely to have good practices [AOR 4.37; 95% CI (1.95–10.21)] compared to those with a negative attitude. Additionally, informal caregivers who demonstrated good awareness regarding HBV infection prevention were three times more likely to have good practices [AOR 3.1; 95% CI (1.4–6.7)] compared to those with poor awareness ( Table 5 ). Section title: Awareness of informal caregivers Educational score: 3.9788551330566406 Domain: biomedical Document type: Study Language: en The prevalence of good awareness in this study was 24.9% (95% CI: 20.7%–29.1%), which is notably lower than reported in previous studies from Ethiopia (44.9%–45.8%) ( 23 ), Nigeria (47.5%) ( 14 ), China (34.6%) ( 24 ), Malaysia (36.9%) ( 13 ), and the USA (32%) ( 9 ). this discrepancy may be attributed to differences in study settings. Previous research was often conducted in community settings with better access to health education, whereas this study focused on informal caregivers in a hospital environment, where the demands of caregiving may limit their engagement with educational activities. Additionally, factors such as lower socioeconomic status and differing cultural attitudes toward health education likely contributed to the lower awareness observed in this study. Section title: Awareness of informal caregivers Educational score: 3.416425943374634 Domain: biomedical Document type: Study Language: en Regarding factors influencing informal caregivers' awareness, informal caregivers of fully functional patients had higher odds of being aware of HBV infection prevention compared to those caring for patients with severe impairments. This finding is consistent with previous studies from the USA ( 9 , 25 ). A possible explanation for this difference is that caregivers of fully functional patients may have better access to information and education, which enhances their understanding and ability to implement preventive measures effectively. Section title: Awareness of informal caregivers Educational score: 2.3247106075286865 Domain: biomedical Document type: Study Language: en Caregivers with a positive attitude toward infection prevention were nearly three times more likely to have good awareness compared to those with a negative attitude, aligning with findings from previous studies in Iran and Malaysia ( 26 , 27 ). This could be because a positive attitude encourages greater engagement with educational resources, leading to improved awareness ( 28 ). Section title: Awareness of informal caregivers Educational score: 3.2184407711029053 Domain: biomedical Document type: Study Language: en Furthermore, Informal caregivers who demonstrated good practices in HBV infection prevention were four times more likely to have good awareness, consistent with studies conducted in Saudi Arabia and Ethiopia ( 29 , 30 ). This could be because caregivers who engage in good practices often have access to educational resources or training that improves their understanding of HBV. Additionally, shared experiences within their communities, where those practicing good infection control serve as role models, may reinforce their awareness. Section title: The practice of informal caregivers Educational score: 4.049981117248535 Domain: biomedical Document type: Study Language: en The prevalence of HBV infection prevention among informal caregivers in this study was 11.6% (95% CI: 8.5–14.7%), which is higher than a similar study conducted in Zambia (0%) ( 18 ). this difference may be due to variations in study settings, periods, and socio-demographic characteristics. However, the prevalence in this study is lower than that reported in previous in Ethiopia 41.5% ( 18 ), 58% in Sudan ( 31 ), and 35% in Nepal ( 32 ). This disparity could be attributed to the fact that earlier studies often involved health professionals, students, or community members with different levels of education, training, and awareness regarding hepatitis B prevention practices, as well as differences in study settings, socio-demographic characteristics, and study periods. Section title: The practice of informal caregivers Educational score: 3.4504144191741943 Domain: biomedical Document type: Study Language: en The odds of practicing HBV infection prevention were higher among informal caregivers who had attained secondary or higher education compared to those with no formal education. This finding aligns with previous studies conducted in Nigeria ( 33 ), Ghana ( 21 ), and Vietnam ( 34 ). Educated informal caregivers are more likely to engage in health-promoting behaviors, including preventive measures against infectious diseases like HBV, and typically have better access to healthcare resources, including information on disease prevention. Section title: The practice of informal caregivers Educational score: 3.3406035900115967 Domain: biomedical Document type: Study Language: en Additionally, the likelihood of good practice was about four times higher among informal caregivers whose patients had acute disease compared to those with chronic disease, which is concurrent with studies conducted in the USA ( 25 , 35 ). The possible reason might be acute illnesses often pose immediate health risks that require rapid responses from caregivers. This urgency can increase awareness and adherence to medical advice, as caregivers are acutely aware of the potential consequences of inaction. Additionally, the emotional impact of an acute diagnosis may drive caregivers to seek more information and follow recommended practices more closely. Section title: The practice of informal caregivers Educational score: 3.0255606174468994 Domain: biomedical Document type: Study Language: en Moreover, informal caregivers with a positive attitude towards HBV infection prevention were more likely to have good practices compared to those with a negative attitude, which is consistent with studies conducted in Addis Ababa, Ethiopia ( 36 ), and Uganda ( 37 ). This can be attributed to the fact that individuals with a positive attitude are generally more motivated. This intrinsic motivation drives them to actively seek out and consistently implement best practices. Additionally, a positive attitude fosters resilience, helping individuals better cope with the stressors and challenges associated with their caregiving responsibilities. Section title: The practice of informal caregivers Educational score: 3.0712029933929443 Domain: biomedical Document type: Study Language: en Lastly, informal caregivers who demonstrated good awareness regarding HBV infection prevention were three times more likely to have good practices compared to those with poor awareness, found to agree with studies conducted in Southeast Asia ( 38 ), and Kenya ( 39 ). This is likely due to their enhanced understanding of the disease, better risk perception, and increased motivation for vaccination and safe practices. Section title: Conclusion Educational score: 3.9434046745300293 Domain: biomedical Document type: Study Language: en This study found that approximately one-quarter of informal caregivers, and only one in eight informal caregivers, had good awareness and practice regarding HBV prevention. Significant factors associated with good awareness included caregivers' attitudes, practices, and the level of physical activity of their patients. Additionally, caregivers' educational status, awareness, attitudes, and the type of disease their patients had were strongly linked to good practices in hepatitis B infection prevention. Section title: Strengths Educational score: 2.295781373977661 Domain: biomedical Document type: Study Language: en The study's strength lies in its ability to offer an accurate snapshot of informal caregivers' awareness and practices within this particular population without requiring long-term follow-up. Section title: Limitations Educational score: 2.348905324935913 Domain: biomedical Document type: Study Language: en The cross-sectional nature of this study makes causal relationships between dependent and independent variables impossible. Section title: Recommendation Educational score: 2.163893461227417 Domain: biomedical Document type: Other Language: en To effectively combat hepatitis B, national policymakers must allocate targeted funding for public health campaigns aimed at raising awareness and promoting prevention strategies, particularly among caregivers. Addis Ababa, the Health Office should organize regular workshops on hepatitis B transmission and prevention, while providing accessible educational materials such as brochures and digital content. Fostering partnerships with local organizations can facilitate community engagement through health fairs and forums. Furthermore, Health professionals should integrate hepatitis B prevention discussions into routine patient care, especially for those with chronic conditions, to educate caregivers. | Review | biomedical | en | 0.999998 |
PMC11695326 | Section title: Exploring neuroplasticity with imaging Educational score: 4.050677299499512 Domain: biomedical Document type: Review Language: en The contributions in this collection reflect diverse approaches to leveraging imaging techniques in rehabilitation research. Caminiti et al. design a protocol to assess the effectiveness of cognitive telerehabilitation methods for mild cognitive impairment, with a focus on evaluating changes in brain connectivity and cognitive performance over multiple follow-up intervals. Kirby et al. explore how sex differences shape white matter neuroplasticity during motor learning. The study shows that females exhibit more pronounced structural adaptations in white matter compared to males, despite similar improvements in motor performance. These findings highlight the importance of considering biological differences when developing personalized rehabilitation strategies. Lotze highlights the importance of performance monitoring in longitudinal fMRI studies to ensure reliable and interpretable results. By addressing variables such as task precision, timing, and physiological responses, the study emphasizes strategies for minimizing confounding factors. These approaches improve the accuracy of imaging data and support the development of more robust neurorehabilitation interventions. Silva et al. investigate the use of Galvanic Vestibular Stimulation (GVS) to improve balance in patients with HTLV-1-associated myelopathy (HAM). The study reports significant short-term improvements in mobility and balance, with patients showing faster mobility (TUG test) and better balance (Berg Balance Scale scores) following the 12-week intervention. However, these gains diminished over time without continued stimulation, suggesting that ongoing use of GVS or home-based devices may be necessary to maintain long-term benefits. Section title: Broad themes and insights Educational score: 4.044543266296387 Domain: biomedical Document type: Review Language: en These studies highlight the complexity of neuroplasticity research and its practical implications for rehabilitation. One of the main takeaways is the importance of personalizing interventions, as demonstrated by Kirby et al. and Caminiti et al. . They show how interventions can be tailored to meet individual needs, whether by considering biological differences or using targeted cognitive telerehabilitation methods. Another critical aspect is the methodological advancements, with Lotze's work on refining performance monitoring in fMRI studies to ensure more reliable and accurate results. Silva et al. also emphasize the importance of long-term strategies for maintaining therapeutic benefits, particularly in chronic conditions like HTLV-1-associated myelopathy, underlining the need for continuous stimulation to sustain progress. Section title: Future directions in rehabilitation science Educational score: 3.966254711151123 Domain: biomedical Document type: Review Language: en The studies in this collection point to promising avenues for advancing rehabilitation science. Caminiti et al. focus on evaluating cognitive telerehabilitation methods for MCI, aiming to understand their effectiveness in improving brain connectivity and cognitive performance. The importance of biological differences, as highlighted by Kirby et al. , suggests that incorporating such factors can lead to more effective, personalized therapies. Lotze's work shows how refining imaging methodologies improves the precision of neuroplasticity studies, further strengthening the foundation of rehabilitation research. Finally, Silva et al. offer insights into the need for continued stimulation to maintain therapeutic gains, pointing to the potential benefits of home-based devices or sustained interventions. | Review | biomedical | en | 0.999996 |
PMC11695328 | Section title: Introduction Educational score: 3.872530221939087 Domain: biomedical Document type: Study Language: en Cardiac arrest during pregnancy is receiving increasing attention ( 1 ). The most common causes of cardiac arrest associated with pregnancy or delivery are described by the American Heart Association as BEAU-CHOPS ( 2 ): bleeding, embolism, aesthetic complications, uterine atony, cardiovascular diseases, hypertension, others, placenta and sepsis. Abnormal uterine bleeding (AUB) is a common symptom and disease in obstetrics and gynecology and increases maternal morbidity and mortality for pregnant women with preexisting AUB-related anemia ( 3 , 4 ). However, there are few reports on cardiac arrest in nonpregnant women caused by AUB ( 5 ). Section title: Introduction Educational score: 2.5469768047332764 Domain: biomedical Document type: Other Language: en Extracorporeal CPR (ECPR) refers to the initiation of cardiopulmonary bypass during the resuscitation of a patient in cardiac arrest. The 2023 American Heart Association Focused Update. Section title: Introduction Educational score: 3.1914222240448 Domain: clinical Document type: Clinical case Language: en The Adult Advanced Cardiovascular Life Support recommends the use of ECPR for patients with cardiac arrest refractory to standard ACLS ( 6 ). In this case, we report ECPR used in a nonpregnant woman with cardiac arrest caused by coronary vasospasm and AUB. Section title: Patient presentation Educational score: 3.953488826751709 Domain: clinical Document type: Clinical case Language: en A 52-year-old female was admitted to the emergency room because of sudden chest pain and chest tightness, with a history of hypertension, coronary heart disease and abnormal uterine bleeding for 20 days. She had multiple previous episodes of chest distress and chest pain after activity that coronary computed tomography angiography showed coronary atherosclerosis. The patient had history of taking aspirin, isosorbide mononitrate sustained-release tablets, atorvastatin, isosorbide mononitrate sustained-release tablets, and stopped aspirin for 1 week because of abnormal uterine bleeding. There's no history of smoking, drug abuse or unprescribed medications. The patient went to the gynecology outpatient service 1 day before the disease's onset. Laboratory examination revealed beta-human chorionic gonadotropin (β-HCG), 0 mIU/ml, and hemoglobin, 70 g/L. Ultrasound revealed multiple myomas of the uterus and uneven thickening of the endometrium. Iron polysaccharide complex capsules were given to correct anemia. Echocardiography was performed 2 days prior, and the left ventricular ejection fraction was 72%. The patient repeatedly experienced chest pain and discomfort at 1 a.m., accompanied by chest tightness and sweating. When she entered the emergency room at 9 a.m., her vital signs were as follows: blood pressure of 128/96 mmHg (right arm, supine position), heart rate of 99 bpm, respiratory rate of 15/min, oxygen saturation of 99% and temperature of 36.6°C. Her cardiac troponin level was totally normal. ECG revealed T wave changes in leads V2, V3, V4, and V5 . Vital sign monitoring, nasal catheter oxygen inhalation (3 L/min), and coronary vasodilator (nicorandil 12 mg intravenous drip) were performed. Considering the AUB, antiplatelet drugs were not given at the first time. Cardiology experts recommend a short-term reexamination of ECG and cardiac troponin to assess the indications for early coronary angiography. The patient's symptoms of chest pain were relieved after treatments. The patient's chest pain reappeared at noon and could not be relieved continuously. During the reexamination of the ECG, the patient suddenly experienced loss of consciousness, sweating, and a decreased heart rate. ECG revealed ST-segment elevation in leads II, III and the AVF which limb leads is type 1 s-degree atrioventricular block (Wenckebach) and chest leads is 2:1 s-degree atrioventricular block . The troponin level was 0.016 ng/ml, which was considered acute myocardial infarction. She was given a loading dose of aspirin 300 mg and clopidogrel 300 mg orally according to the cardiology consultation. Bedside ultrasound revealed that left ventricular systolic function decreased diffusely to less than 10%, and the femoral artery was untouched. Endotracheal intubation, isoproterenol, and norepinephrine were given immediately. After that, the patient underwent episodes of cardiac arrest with pulseless electrical activity, and ECPR was started after traditional cardiopulmonary resuscitation. The ICU team placed VA-ECMO beside the bed to restore spontaneous circulation. The vital signs were as follows: blood pressure of 117/95 mmHg (right arm, supine position, 0.4 µg/kg/min norepinephrine), heart rate of 131 bpm, controlled ventilation at 14/min, and a temperature of 36.1°C. The initial setting parameters of ECMO were as follows: rotation speed of 2,780 r, flow rate of 3.63 L/min, gas flow rate of 3 L/min, oxygen concentration of 50%, and water tank temperature of 36°C. The blood gas analysis showed that the patient's hemoglobin had dropped to 49 g/L at 3:40 p.m. and we gave blood transfusion immediately at 4 p.m. The patient underwent urgent coronary angiography. Figure 3 revealed a diffuse slenderness of the coronary artery and severe narrowing of the left circumflex (LCX) arteries, in which the heaviest stenosis was 90%. Suspecting spasm, the left coronary artery was injected intracoronary with 100 µg nitroglycerine, which completely relieved the spasm. After relief of spasm, angiography showed left anterior descending artery (LAD) had atherosclerosis with no significant stenosis or occluding lesions, which in good blood flow. The inferior myocardial infarction was confirmed to be caused by coronary vasospasm, and the postoperative electrocardiogram revealed that the ST segment returned to baseline. The cardiac troponin levels obtained 4 and 12 h later were 2.1 ng/ml and 2.2 ng/ml, respectively. Section title: Patient presentation Educational score: 3.908582925796509 Domain: clinical Document type: Clinical case Language: en After the patient entered the ICU, the central venous pressure (CVP) was 9 mmHg. The arterial blood gas test results revealed a pH of 7.32, PaCO2 of 26.1 mmHg, PaO2 of 122 mmHg, and lactic acid level of 8.5 mmol/L. The differential pressure of arterial and central venous carbon dioxide was 3.4 mmHg, and the central venous oxygen saturation was 54%. On the one hand, we strengthened sedation and analgesia and implemented target temperature management to reduce oxygen consumption. On the other hand, the patient received a blood transfusion to increase the oxygen supply. On the second day after the operation, blood gas analysis revealed that the pH, central venous oxygen saturation and lactic acid levels stabilized and improved. During ECMO treatment, the vasoactive agents controlled the mean arterial pressure above 75 mmHg, and the level of lactic acid decreased and returned to normal. The results of the hemodynamic monitoring of the patient during ECMO support are shown in Table 1 . The patient was given 100 mg/d aspirin, 20 mg/n atto vastatin, 10 mg/d ezetimibe, 24 mg/h nicorandil, 50 mg/d isosorbide mononitrate, and 90 mg/d diltiazem. As the patient experienced a decreasing trend in her cardiac troponin level, she had no recurrent chest pain, and her left ventricular ejection improved to 55%. With improvements in cardiopulmonary conditions, the number of ECMO settings gradually decreased. ECMO support was removed at 4 days after admission, and weaning from mechanical ventilation was successful after 5 days. Section title: Patient presentation Educational score: 3.2513108253479004 Domain: clinical Document type: Clinical case Language: en The patient experienced intermittent vaginal bleeding, and the amount of vaginal bleeding was approximately 50–600 ml per day. The patient was diagnosed with adenomyosis, and the hemoglobin level returned to 105 g/L after treatment. However, there was no tangible effect after intramuscular injection of 10 U oxytocin and infusion of red blood cells, with hemoglobin decreasing to 77 g/L. Uterine artery embolization was performed on the 8th day, and the amount of vaginal bleeding decreased significantly after the operation. Hemoglobin increased to 90 g/L after the operation. After 13 days, with no recurrent chest pain and in stable condition, the patient was transferred from the ICU to the regular cardiology ward and discharged 21 days after the operation. Section title: Discussion Educational score: 4.003457069396973 Domain: clinical Document type: Clinical case Language: en Here, we describe a case of cardiac arrest due to coronary vasospasm and AUB. She had recurrent chest pain and tightness, but no abnormalities were detected via electrocardiogram, echocardiography, or myocardial injury markers when symptoms did not occur. The patient subsequently suddenly experienced cardiac arrest and was able to regain spontaneous circulation with VA-ECMO support. Coronary angiography confirmed diffuse spasms in multiple coronary arteries. Therefore, we believe that coronary vasospasm and type 2 myocardial infarction caused by anemia were the main causes of cardiac arrest in this patient. Section title: Discussion Educational score: 2.776876926422119 Domain: biomedical Document type: Clinical case Language: en To the best of our knowledge, there are few reports on cardiac arrest in nonpregnant women caused by AUB. Takeshi Yagi reported a 57-year-old woman who experienced cardiac arrest due to massive hemorrhage from uterine adenomyosis with leiomyoma ( 5 ). The author suggested that cardiac arrest was caused by hemorrhagic shock. Section title: Discussion Educational score: 4.212700843811035 Domain: biomedical Document type: Study Language: en In patients with known or presumed CAD, acute stressors such as bleeding, tachyarrhythmia, hypoxia or hypotension may result in myocardial injury. According to the fourth universal definition of myocardial infarction, the pathophysiological mechanism leading to ischemic myocardial injury in the case of a mismatch between oxygen supply and demand has been classified as type 2 myocardial infarction ( 7 ). The myocardial oxygen supply/demand imbalance attributable to acute myocardial ischemia may be multifactorial and related either to reduced myocardial perfusion due to fixed coronary atherosclerosis without plaque rupture, coronary vasospasm, coronary microvascular dysfunction, coronary embolism, coronary artery dissection with or without intramural hematoma or other mechanisms that reduce the oxygen supply or increase the myocardial oxygen demand ( 8 ). Section title: Discussion Educational score: 3.8091800212860107 Domain: biomedical Document type: Other Language: en It appears advisable in the acute setting to treat the underlying ischemic imbalance of oxygen supply and demand. This treatment may include volume adjustment, blood pressure management, the administration of blood products, heart rate control, and respiratory support ( 9 , 10 ). In this case, the patient experienced recurrent symptoms of chest pain that culminated in cardiac arrest, possibly related to anemia that was not corrected in time. Section title: Discussion Educational score: 3.930713415145874 Domain: biomedical Document type: Review Language: en Extracorporeal cardiopulmonary resuscitation (ECPR) is a resuscitation mode that can effectively improve the success rate of cardiopulmonary resuscitation in patients with cardiopulmonary arrest, especially in-hospital cardiopulmonary arrest. In the treatment of cardiac arrest patients, the spontaneous circulation recovery rate is as high as 95% ( 11 ), and the discharge survival rate is as high as 27.6%–50% ( 11 – 13 ). The 2023 American Heart Association Focused Update on Adult Advanced Cardiovascular Life Support recommends the use of ECPR for patients with cardiac arrest refractory to standard ACLS ( 5 ). Section title: Discussion Educational score: 2.201808214187622 Domain: biomedical Document type: Other Language: en There are currently no data on the incidence and mortality of myocardial infarction induced by AUB. Therefore, type 2 myocardial infarction caused by abnormal uterine bleeding may be underrecognized. In the treatment of cardiac arrest caused by type 2 myocardial infarction, it is very important to correct predisposing factors such as anemia as soon as possible. | Study | biomedical | en | 0.999997 |
PMC11695329 | Section title: Introduction Educational score: 4.514013290405273 Domain: biomedical Document type: Review Language: en Allostatic Load (AL) describes the wear and tear resulting from physiological responses upon chronic stress exposure . As an index composed of inflammatory, metabolic, and cardiovascular biomarkers , AL summarises a global level of systemic dysfunction and has been associated with an increased risk of developing several pathological conditions including major depression, frailty, changes in brain volume, cardiovascular disease, chronic fatigue syndrome, fibromyalgia, diabetes type 2, seizures, breast cancer, cognitive impairment, and Alzheimer’s disease (AD) . Various studies investigated factors associated with AL, from psychological to sociodemographic but in general, high levels of AL can result from three situations . The first, and most known, is the repeated exposure to stressors that leads to a chronic enhancement of physiological arousal. This situation refers to both the number and frequency of stressors experienced through life. The second situation refers to the inability to successfully adapt to adversity and stress demands through effective coping strategies. These are reflected in the concept of resilience and can be considered as protective factors against allostatic overload . A third situation occurs when the stress response cannot be terminated after the end of the stressor exposure. Here, the physiological arousal triggered by fear or danger is unable to be extinguished and is usually re-experienced when exposed to cues or contexts related to the event, as seen in patients and rodent models of post-traumatic stress disorder (PTSD) . Thus, an event that occurred a long time ago can still exert its arousing influence many years later to cause increased AL levels. Section title: Introduction Educational score: 4.096584796905518 Domain: biomedical Document type: Study Language: en Besides resilience, lifestyle and habits have also been studied to identify protective factors against allostatic overload . Among them, healthy dietary habits have been consistently associated with lower AL in both young and older adults . Particularly, adherence to a Mediterranean diet has been frequently related to lower cardiovascular burden , which is a strong component of the AL index . Similarly, regular engagement in physical activity has been associated with lower levels of AL in European, Latin-American and Asian populations , with an effect not restricted to vigorous sports but also to mild and moderate occupational and leisure activities . Cognitive habits, such as reading, practicing another language, painting, or playing an instrument, have also been found to be protective against stress exposure and associated with lower levels of AL . In addition, sleep has received special attention in relation to AL and stress exposure. While poor sleep quality has been found to be associated with higher levels of AL , evidence also reports impaired sleep upon stress exposure and has been identified as a key symptom of PTSD . Section title: Introduction Educational score: 4.094099044799805 Domain: biomedical Document type: Study Language: en In the present study, we analysed how lifetime stressors, and a healthy lifestyle indicated by adherence to a healthy diet cognitive engagement and physical activity modulate AL in a British cohort of cognitively normal mid-life adults. By including the self-reported perceived influence of the stressor events and the levels of resilience as potential mediators, we evaluated if the modulation depends also on the ability to cope and overcome the allostatic demands. Following the extensive evidence of the bidirectional relation between stress and sleep quality, we further assessed its role in the modulating effects of AL. Section title: Participants Educational score: 2.496417760848999 Domain: biomedical Document type: Study Language: en Data from the PREVENT study [v700 baseline dataset ] were used. As described previously , the PREVENT cohort recruited mid-life participants (age: 40–59 years) from sites in Edinburgh, West London, Dublin, Cambridge and Oxford. All participants provided written informed consent before participation and were free of cognitive impairment at the baseline visit. Section title: Ethics Educational score: 1.0990431308746338 Domain: other Document type: Other Language: en Multi-site ethical approval was granted by the UK London-Camberwell St Giles National Health Service (NHS) Research Ethics Committee , which operates according to the Helsinki Declaration of 1975 . Separate ethical approval was received for the Dublin site, from Trinity College Dublin School of Psychology Research Ethics Committee and the St James Hospital/Tallaght University Hospital Joint Research Ethics Committee. All substantial protocol amendments have been reviewed by the same ethics committees with a favourable opinion granted before implementation at sites. Section title: Ethics Educational score: 1.5993045568466187 Domain: biomedical Document type: Other Language: en All necessary participant consent has been obtained before assessments and the appropriate institutional forms have been archived. Any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients, or participants themselves) outside the research group so cannot be used to identify individuals. Participants in the study remained anonymous, identifiable information was held at site and only accessible by the direct research team. This identifiable information was not shared to the study data base where each participant was only identified by a study ID number. Section title: Biomarkers collection Educational score: 4.127590656280518 Domain: biomedical Document type: Study Language: en Blood samples were collected in a fasted state during the baseline visit and analysed immediately at local laboratories analysed in local laboratories for biochemistry and haematology measures using NHS standard procedures . Vital signs were collected after breakfast by trained members of the research team. Blood pressure and heart rate were collected in triplicate (both supine and standing) and a mean of the three measures. Height and weight were recorded for body mass index (BMI) calculation and measurements of waist and hip circumference were documented. Fourteen biomarkers were assessed for inflammatory/immune (creatinine, albumin, C-reactive protein [CRP], fibrinogen), cardiovascular (systolic blood pressure [SBP], diastolic blood pressure [DBP], resting heart rate [RHR], and waist-to-hip ratio [WHR]), and metabolic (total cholesterol, high-density-lipoprotein [HDL] cholesterol, low-density-lipoprotein [LDL] cholesterol, glycemia, triglycerides, and BMI) systems. Section title: Comprehensive AL score (ALCS) Educational score: 4.0882110595703125 Domain: biomedical Document type: Study Language: en All biomarkers were scored to create a comprehensive AL index (ALCS), as previously described . Initial categories for “no-risk” (zero points), “at-risk” (one point), and “high-risk” (two points) were defined for each biomarker, based on both clinical thresholds ( National Institute for Health and Clinical Excellence (NICE), 2006 ; Fuggle, 2018 ) and quartiles from sex-specific distributions. When the clinical upper limit (clinical-up) was higher than the 75th percentile (p75: creatinine, triglycerides, CRP, SBP, DBP), the at-risk category was defined between ≥p75 – ≤clinical-up (no-risk: <p75 and high-risk: >clinical-up). When the clinical upper limit was lower than p75 (total cholesterol, LDL cholesterol, BMI, WHR), at-risk was defined between ≥clinical-up – ≤p75 (no-risk: <clinical-up, high-risk: >p75). For reverse biomarkers (albumin, HDL cholesterol), if the clinical lower limit (clinical-low) was below the 25th percentile (p25), at-risk was defined as ≤ clinical-low – ≥p25 (no-risk: >p25 and high-risk: <clinical-low). For RHR, only clinical categories provided by the British Cardiovascular Society for age and sex were used. Section title: Comprehensive AL score (ALCS) Educational score: 3.9774394035339355 Domain: biomedical Document type: Study Language: en Medication treatments coded through the Anatomic Therapeutic Chemical (ATC) classification system were scored as high-risk to account for potential masking some biomarkers values, as follows: total cholesterol, triglycerides and LDL for lipid modifying agents (C10); systolic and diastolic blood pressure for anti-hypertensive medication (C02, C03, C09); resting heart rate for beta-blockers (C07) or calcium blockers (C08); and glycemia for insulin or analogues (A10). Section title: Comprehensive AL score (ALCS) Educational score: 2.48313307762146 Domain: biomedical Document type: Study Language: en The summed scores were used as continuous variable for the mediation analysis. The decision algorithm is described in Supplementary Figure S1 , and clinical thresholds and quartiles values are detailed in Supplementary Table S1 . Section title: Life stressors Educational score: 4.1394147872924805 Domain: biomedical Document type: Study Language: en Stressful life events were assessed through the self-reported Life Stressor Checklist—Revised (LSC-R) , where participants are asked if they had experienced a set of 30 stressors across their lives, such as natural disasters, sexual assault, death of a relative, divorce, etc. Participants also report their age at the time of the event, age when the event ended, belief that they were in harm (“yes” or “no”), feelings of helplessness (“yes” or “no”), and the perceived influence of the event over the past year (rated on a five-point intensity scale from 1 = “not at all or never” to 5 = “extremely”) . Items 29 and 30 are open questions to identify other stressors not listed before, so they were excluded from the current analysis as many answers were found to replicate the same events listed previously, leaving a maximum total score of 28 possible life stressors. Traumatic events were identified following the definition proposed by the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) criteria A for post-traumatic stress disorder (PTSD) diagnosis, as an exposure by direct experience, witnessing or learning, to actual or threatened death, serious injury, or sexual violence . Similarly, the International Classification of Diseases 11th Revision (ICD-11) defines stressful events as an extremely threatening situation, including “ natural or human-made disasters, combat, serious accidents, torture, sexual violence, terrorism, assault.… witnessing the threatened or actual injury or death of others in a sudden, unexpected, or violent manner; and learning about the sudden, unexpected or violent death of a loved one ” . In these proposals, events not involving an immediate threat to life or physical injury such as divorce or job loss are considered psychosocial stressors, and medical incidents involving natural causes, such as a heart attack, are not considered traumatic . Thus, items 1–3, 12, 13, 17, 19–23, 25–28 were classified as traumatic stressors, and items 4–11, 14–16, 18, 24 were classified as psychosocial stressors (description of items in Supplementary material S1 ). Section title: Pyramid score Educational score: 3.977447509765625 Domain: biomedical Document type: Study Language: en The Pyramid score is a widely used scoring algorithm to evaluate adherence to a Mediterranean-style diet . Each contributing food component was coded on a continuous scale of 0–1 with a total possible score of 15 points and was calculated as previously described . Briefly, scores were derived from the Scottish Collaborative Group Food Frequency Questionnaire (SCG-FFQ), which gathered data on 175 different foods and drinks consumed by participants over the last two to three months. Total energy intake (kcal/day) was derived from the dataset and included in the analysis. Participants with extreme energy intakes (<600 kcal, >6,000 kcal) were excluded from the analysis (for full details see Supplementary Table S2 ). Section title: Pyramid score Educational score: 2.834243059158325 Domain: biomedical Document type: Study Language: en From the 620 participants selected with complete data for AL scoring, 570 had sufficient data to calculate a Pyramid score. To be able to obtain modification indices for structural equation models (SEMs) and estimate indirect effects via bootstrapping, missing values for the remaining 50 participants were imputed by a regression imputation. Section title: Cognitive and sports habits Educational score: 3.6239964962005615 Domain: biomedical Document type: Study Language: en Healthy habits related to physical or cognitive activities were evaluated through selected items from the Lifetime of Experiences Questionnaire (LEQ) . Participants are asked to report on average, how often they take part in each activity (rated on a six-point intensity scale from 0 = “never” to 5 = “daily”) from the age of 30 until the end of their working life or present (if still in work). Cognitive habits were calculated by summing the scores for items related to the play/practice a musical instrument, artistic past-time, reading and practicing a second language, whereas sports habits summarised scores related to the practice of mild, moderate, or vigorous physical activities. Full item descriptions are detailed in Supplementary material S2 . Section title: Perceived influence of stressors over the past year Educational score: 2.640183448791504 Domain: biomedical Document type: Study Language: en To discriminate between the effects derived from the number of life stressors and their actual impact at the time of evaluation, scores of the self-perceived influence over the past year obtained by the LSC-R were derived separately for traumatic and psychosocial stressors, and included as a mediator between the exogenous variables and AL. Section title: Resilience Educational score: 2.990243434906006 Domain: biomedical Document type: Study Language: en As a potential protective factor mediating the effect of stressors on AL, an index of self-reported resilient attitudes was included through the Connor-Davidson Resilience Scale (CDRS) . This provides an overall index for self-reported resilient attitudes through a 25-item Likert scale, with points ranging from 0 to 4, and higher scores reflecting greater resilience. Section title: Poor sleep quality Educational score: 3.8644769191741943 Domain: biomedical Document type: Study Language: en Self-reported sleep quality was assessed through the Pittsburgh Sleep Quality Index (PSQI) questionnaire . It summarises seven components of sleep quality (self-perceived quality, latency, duration, efficiency, disturbance, medications, and daytime dysfunction) in a total score ranging between 0 and 21, with higher scores reflecting poorer sleep quality. Section title: Demographic covariates Educational score: 1.7255654335021973 Domain: biomedical Document type: Study Language: en Demographic covariates included age, sex (categorical, coded as males = 1, females = 2), and years of education. Section title: Structural equation models for assessing mediation path analysis Educational score: 3.9343249797821045 Domain: biomedical Document type: Other Language: en Structural equation models (SEMs) path analysis is a generalization of multiple regression procedures that allows to include multiple endogenous (dependent) variables, to impose restrictions on one or more parameters (i.e., set a parameter to zero, set two parameters to be equal, or restrict the assessment of unnecessary regressions), and to assess indirect effects through chained associations between an exogenous (independent) variable and mediating endogenous variables . The latter is crucial when no direct associations between a predictor and an outcome are found but can be theoretically hypothesised to be mediated indirectly through an additional association with another variable between them. Section title: Structural equation models for assessing mediation path analysis Educational score: 4.042056083679199 Domain: biomedical Document type: Study Language: en Based on the reviewed evidence, we developed a conceptual framework for SEM path analyses with the following exogenous variables: (i) Experiential factors , including the number of stressors (traumatic or psychosocial), Pyramid score, cognitive habits, and sport habits; and (ii) Demographic factors , including sex, age, and years of education covariates. In the first model (Model 1), we evaluated three a priori potential mediating pathways that may play a role in the associations between the influencing factors and AL: (i) through the perceived influence of the stressors; (ii) through resilience; and (iii) through a chain mediation of resilience on the perceived influence . Section title: Structural equation models for assessing mediation path analysis Educational score: 4.1005096435546875 Domain: biomedical Document type: Study Language: en Following the results of the first model, and according to the theoretical evidence reviewed, sleep quality was included as a third mediator between the first two (perceived influence and resilience) and AL (Model 2), adding three new potential mediating pathways to the previously described: (iv) sequentially through perceived influence to sleep; (v) through resilience to sleep; and (vi) through resilience to perceived influence to sleep . No direct effects paths from traumatic or psychosocial stressors to poor sleep quality were specified, as evidence reports that the effects of stressor exposure on sleep does not follow a uniform dose–response relationship but can vary between individuals, and additional factors such as stressor chronicity, resilience ability and the event appraisal need to be accounted to estimate the impact of differential sleep impact in response to stress . Therefore, we hypothesised than the mediating effect of sleep between the exogenous variables and AL had to arise from a previous indirect effect via resilience, perceived influence or resilience to perceived influence. Section title: Statistical analysis Educational score: 3.6122705936431885 Domain: biomedical Document type: Study Language: en All analyses were conducted using IBM SPSS Statistics v.27.0 and Amos (Analysis of Moment Structures) v.27.0 . Statistical differences between sex for all demographic and behavioural variables were evaluated by two-sided Mann Whitney-U rank sum tests, as normality estimated by Shapiro–Wilk tests were below the rejection value of <0.05 (details in Supplementary Table S3 ). Section title: Statistical analysis Educational score: 3.0977227687835693 Domain: biomedical Document type: Study Language: en Pearson’s product moment correlation coefficients were computed for preliminary evaluation of bivariate associations among all the studied variables. Section title: Model identification Educational score: 2.3700945377349854 Domain: biomedical Document type: Study Language: en In order to conduct SEM path analysis, identification of the hypothesised path models was checked through the compliance with the T-rule (necessary but not sufficient) and the recursive rule (sufficient but not necessary) . Section title: Model identification Educational score: 3.8432624340057373 Domain: biomedical Document type: Study Language: en The T-rule states that for a model to be sufficiently identified, the number of known parameters (calculated as k *( k + 1)/2 + k , where k is the number of observed exogenous and endogenous variables) must be equal to (just-identified model) or greater than (over-identified model) the number of free parameters that needs to be estimated. All the evaluated models complied with the rule for over-identification (Model 1, Traumatic stressors: known = 65 > estimated = 58; Model 1, Psychosocial Stressors: known = 65 > estimated = 59; Model 2, Traumatic stressors: known = 77 > estimated = 63; Model 2, Psychosocial Stressors: known = 77 > estimated = 63). Section title: Model identification Educational score: 2.3353660106658936 Domain: biomedical Document type: Study Language: en Thus, all assessed models were over-identified and recursive, given no correlated residuals, bi-directional effects, or feedback loops were included. Section title: Normality assumption Educational score: 4.2087602615356445 Domain: biomedical Document type: Study Language: en As shown by the use of Mann Whitney-U rank sum tests for males vs. females comparisons ( Supplementary Table S3 ) all assessed variables showed deviations from normality (assessed by Shapiro–Wilk test). However, for sample sizes greater than 300, either an absolute skew value larger than 3 or an absolute kurtosis (proper) larger than 8 may be used as reference values for determining substantial non-normality . Both skewness and kurtosis values for each variable were assessed and reported in Supplementary Table S4 , with only influence of traumatic events showing a significant deviation from kurtosis normality (kurtosis = 8.89). However, all multivariate normality Mardia’s coefficients calculated for each SEM model showed significant deviations from normality (details in Supplementary Table S4 ). For the purpose of SEM analysis, only normality for the endogenous variables (AL, sleep quality, resilience and perceived influence) is required for accurate estimation of Maximum Likelihood, and violations of normality can both inflate likelihood-ratio χ 2 tests—leading models to be rejected more often than they should—and underestimate standard errors—increasing the probability of error type I when testing significance of individual parameters . To address this violation, a bootstrapping approach was used, as one of the most recommended for samples large enough to be representative where univariate and multivariate normality assumptions are not met . The bootstrapping procedure consists in generating multiple new samples—at least 1,000 —from the original database, to construct a bootstrap sampling distribution that will operate in a similar way as those traditionally associated with parametric inferential statistics (i.e., t or F distributions), but without the need to meet the normality assumption for an adequate estimation of parametric values, such as regression coefficients . As this sample-based distribution is drawn through consecutive replacements of the original sample, it allows to generate bias-corrected standard errors and confidence intervals for an accurate estimation of statistical significance, correcting the increased probability of error type-I caused by the violation of normality. Therefore, a 1,000-samples bootstrapping was performed for all the SEM models analysed, with bias-corrected confidence intervals (CI) set at 95%. Only bias-corrected standard errors, CI, and adjusted p -values are reported for direct and indirect effects. Section title: Linearity assumption and collinearity assessments Educational score: 4.114161491394043 Domain: biomedical Document type: Study Language: en Linearity assumption was checked on each pair of associations between dependent and independent variables analysed in the models. Deviation from linearity is calculated on SPSS as follows: after subtracting the within groups form residuals sum of squares, difference is divided by degrees of freedom (df) of residuals—df within group to the deviation mean squared. An F value is computed as the mean square ratio of deviation/within groups and the p value is calculated with the corresponding degrees of freedom (df deviation, df withing groups). p -values <0.05 indicate a significant deviation from linearity. No violations of linearity were found on any relation between dependent and independent variables assessed by the models, as shown in Supplementary Table S5 . Section title: Linearity assumption and collinearity assessments Educational score: 4.012104511260986 Domain: biomedical Document type: Study Language: en The presence of multicollinearity was checked by performing a multiple regression analysis on each of the dependent variables assessed in the SEM models, with the corresponding independent variables as covariates. Variance inflation factor (VIF) >5 and tolerance statistic <0.2 were considered as indicators of multicollinearity. Results are summarised in Supplementary Table S5 , showing no collinearity effects. Section title: Imputation of missing values of the Pyramid score variable Educational score: 4.149141788482666 Domain: biomedical Document type: Study Language: en To obtain modification indexes, bias-corrected standard errors, CI, and adjusted p-values through bootstrapping procedures in the fitted SEM models, the full dataset is required to be complete. To choose the best procedure to deal with the 50 missing values of the Pyramid score variable, the missing data mechanism operating needed to be determined. Although there is no formal testing to assess the missing data mechanism that is present in the data, we performed a logistic regression to assess for the plausibility of either a missing at random (MAR) or missing completely at random (MAR) assumptions . In brief, if significant relations between missing Pyramid Score data and all the rest of the variables in the dataset were found, then MAR could be assumed. If no relationships are shown, then MCAR can be concluded. The possibility of missing not-at random (MNAR) assumption was discarded, as it implies that missing data is systematically related to events or factors that were not measured in the study, which cannot be plausible when only an 8.1% of data is missing for one variable . Therefore, after transforming missing and non-missing Pyramid scores into dichotomic categories, where 0 corresponded to non-missing values and 1 corresponded to missing values, a logistic regression on the full dataset was performed setting non-missing (values = 0) as reference category. Given no significant associations were found between the missing data and any the variables (see full report in Supplementary Table S6 ), we concluded that it was possible to assume that missing data were MAR, and hence, assumptions for performing imputation were satisfied. Section title: Imputation of missing values of the Pyramid score variable Educational score: 4.124351978302002 Domain: biomedical Document type: Study Language: en Robust Full Information Maximum Likelihood (RFIML) imputation method was then performed on AMOS, as suggested by previous research to obtain better results with MCAR data and to allow for bias-correction to deal with non-normal data . In brief, after fitting the model using the full information to calculate maximum likelihood, model parameters are set equal to their maximum likelihood estimates and linear regression is used to predict the unobserved values for each case as a linear combination of the observed values for that same case . The advantage of using this method for complex SEM path analysis is that it considers all the relations assessed in the model instead of relying only on one particular set of associations with the variable to be predicted . Section title: Assessments of goodness of fit and modification indices Educational score: 4.090456008911133 Domain: biomedical Document type: Study Language: en Hypothesised structural models were evaluated for goodness of fit by the following indices and criteria : Likelihood-ratio χ 2 test ( p ≥ 0.05), Comparative Fit Index (CFI ≥ 0.95) and Tucker-Lewis Index (TLI ≥ 0.95), the root-mean-square error of approximation (RMSEA<0.06), and the standardised root mean squared residual (SRMR<0.08). If an initial model assessment showed poor fit for the χ 2 test ( p < 0.05), calculated modification indices (MI) were considered for post-hoc model modifications. The suggested parameters to add for model improvement were selected from MI values greater than 3.84 as indication of significant improvement , and if they were consistent with the theoretical construct. Section title: Assessments of goodness of fit and modification indices Educational score: 2.634411334991455 Domain: biomedical Document type: Study Language: en Direct effects estimates were estimated as standard regression coefficients, whereas total effects were estimated but not considered for analysis due to the multiple pathways and mediations involved might lead to confounding interpretations. Section title: Assessments of goodness of fit and modification indices Educational score: 3.9982593059539795 Domain: biomedical Document type: Study Language: en For evaluation of total indirect effects of the mediating pathways of interest , products of the standardised coefficients from the sequential mediation paths were labelled and set as user-defined estimands on AMOS. standardised estimates and p -values were calculated by bootstrapping, with 1,000 bootstrap samples, and bias-corrected percentile method, with confidence intervals at 95%. All significant effects were set at α = 0.05. Section title: Demographic and behavioural characteristics Educational score: 3.977437734603882 Domain: biomedical Document type: Study Language: en The analytical sample included 620 participants from the PREVENT dementia study (61.13% females), with an average age of 51.3 (SD = 5.48) years old (females: 50.97, SD = 5.41), and a mean of 16.62 (SD = 3.44) years of education. Demographic and behavioural characteristics of the PREVENT participants included in the analyses are detailed in Table 1 . Comparisons between sexes revealed higher AL scores and greater engagement in sports in males. Females showed higher Pyramid scores and an increased number and perceived influence of psychosocial stressors. No differences were found between males and females for age, years of education, number and perceived influence of traumatic stressors, resilience, and sleep quality (details in Supplementary Table S3 ). Section title: Bivariate correlations Educational score: 3.9943013191223145 Domain: biomedical Document type: Study Language: en As shown in Figure 2 and Supplementary Table S7 detailing bivariate correlations results, small to moderate significant correlations were found between all variables assessed, except for two very strong correlations between number of stressors and their corresponding perceived influence scores (traumatic r = 0.828 and psychosocial r = 0.823, respectively). Among demographic covariates (see full details in Supplementary Table S7 ), small significant correlations were found in males between AL and sport habits, and in females between the Pyramid score, psychosocial stressors, and their perceived influence. Age showed a weak negative correlation to years of education and positive to higher AL, whereas higher education was weak but significantly correlated to lower AL, the Pyramid score and engagement in cognitive activities. Section title: Bivariate correlations Educational score: 3.761052370071411 Domain: biomedical Document type: Study Language: en Significant but weak correlations were found between AL and the number of traumatic ( r = 0.152) and psychosocial stressors ( r = 0.142), their respective perceived influence (traumatic r = 0.155 and psychosocial r = 0.158), poor sleep quality ( r = 0.161), smaller pyramid diet scores ( r = −0.220) and sports habits ( r = −0.217). Section title: Bivariate correlations Educational score: 3.7319281101226807 Domain: biomedical Document type: Study Language: en The number of traumatic stressors was weakly correlated with cognitive habits ( r = 0.110) and poor sleep quality ( r = 0.152) and had a moderate correlation to psychosocial stressors ( r = 0.444) and their perceived influence ( r = 0.397). Similarly, psychosocial stressors were moderately correlated to the perceived influence of traumatic stressors ( r = 0.478), and weak but significantly correlated to cognitive habits ( r = 0.143), poorer sleep quality ( r = 0.182), and less engagement in sport habits ( r = −0.146). Section title: Bivariate correlations Educational score: 3.8557724952697754 Domain: biomedical Document type: Study Language: en Higher Pyramid scores were correlated with both cognitive and sports habits ( r = 0.172 and r = 0.125, respectively), whereas more frequent engagement in cognitive activities was correlated with a higher perceived influence of both traumatic ( r = 0.125) and psychosocial stressors ( r = 0.159). Sports habits showed weak but significant correlations to higher levels of resilience ( r = 0.160), better sleep quality ( r = 0.120) and lower perceived influence of psychosocial stressor ( r = −0.132), while poor sleep quality was correlated with higher influence of traumatic ( r = 0.255) and psychosocial stressors ( r = 0.230), and lower resilience levels ( r = −0.255). Section title: Effects of life stressors and healthy habits on AL mediated by perceived influence and resilience Educational score: 4.117574214935303 Domain: biomedical Document type: Study Language: en To assess the association of stressful life events and healthy habits on AL, SEMs were estimated separately for traumatic and psychosocial stressors, with perceived influence over the last year and resilience as mediators. The traumatic stressors model shows satisfactory fit statistics ( χ 2 (7) = 7.448, p = 0.384; TLI = 0.997, CFI = 1.00, RMSEA = 0.01, SRMR = 0.0168). For the psychosocial stressors model an additional direct path from years of education to AL was included to improve model fit (modification index = 4.345; model fit: χ 2 (6) = 10.83, p = 0.094; TLI = 0.963, CFI = 0.995, RMSEA = 0.036, SRMR = 0.0231). Section title: Effects of life stressors and healthy habits on AL mediated by perceived influence and resilience Educational score: 4.120326995849609 Domain: biomedical Document type: Study Language: en Figure 3A shows the path diagram of the modulation of AL by traumatic stressors and healthy habits by mediation of perceived influence and resilience. Significant direct effects on AL were found from Pyramid scores ( β = −0.171, p = 0.002, 95% CI = [−0.239,-0.093]) and sports habits ( β = −0.189, p = 0.002, 95% CI = [−0.269,-0.111]). The latter also exerted a significant direct effect on resilience ( β = 0.152, p = 0.003, 95% CI = [0.061, 0.234]). Significant direct effects were also found from resilience to perceived influence ( β = −0.042, p = 0.036, 95% CI = [−0.085,-0.004]), but not from perceived influence on AL ( β = −0.103, p = 0.203, 95% CI = [−0.067, 0.238]) or from resilience to AL ( β = 0.001, p = 0.982, 95% CI = [−0.07, 0.076]). None of the selected mediation paths revealed significant indirect effects (Full statistical details described in Supplementary Table S8 ). Section title: Effects of life stressors and healthy habits on AL mediated by perceived influence and resilience Educational score: 4.122767448425293 Domain: biomedical Document type: Study Language: en The assessment of psychosocial stressors through Model 1 showed equivalent direct effects of diet ( β = −0.156, p = 0.001, 95% CI = [−0.231, −0.08]) and sports habits ( β = −0.178, p = 0.003, 95% CI = [−0.254, −0.096]) on AL, and from sports habits to resilience ( β = 0.148, p = 0.003, 95% CI = [0.049, 0.229]). Significant direct effects were also found from resilience to perceived influence ( β = −0.088, p = 0.001, 95% CI = [−0.137, −0.039]), but not from perceived influence on AL ( β = 0.141, p = 0.07, 95% CI = [−0.015, 0.299]) or from resilience to AL ( β = 0.013, p = 0.739, 95% CI = [−0.065, 0.087]). A partial mediation on AL by sports habits was revealed by a significant indirect effect ( β = −0.002, p = 0.027, 95% CI = [−0.006, 0]) through the third analysed pathway ( ←Resilience ←perceived influence ← AL ). Full statistical details are summarised in Supplementary Table S9 . Section title: Sleep quality as pivotal mediator between life stressors and healthy habits on AL Educational score: 4.132770538330078 Domain: biomedical Document type: Study Language: en Given that the analyses conducted through Model 1 did not reveal any direct or mediated associations between number of stressors and AL, we hypothesised that an additional factor could be underlying this effect. According to the reviewed evidence, sleep quality was chosen as an additional mediator between the effects of both perceived influence and resilience on AL to conduct a further assessment. In this second model, we hypothesised that the effect of life stressors and healthy habits mediated by perceived influence and resilience would modulate AL by affecting sleep quality. Satisfactory fit statistics were achieved for the sleep-modulated models of traumatic stressors ( χ 2 (14) = 17.385, p = 0.236; TLI = 0.987, CFI = 0.997, RMSEA = 0.02, SRMR = 0.0193) and psychosocial stressors ( χ 2 (14) = 18.999, p = 0.165; TLI = 0.981, CFI = 0.995, RMSEA = 0.024, SRMR = 0.0253). Section title: Sleep quality as pivotal mediator between life stressors and healthy habits on AL Educational score: 4.138701915740967 Domain: biomedical Document type: Study Language: en For traumatic stressors , the SEM analysis confirmed significant associations between Pyramid scores and sports habits with AL ( β = −0.176, p = 0.002, 95% CI = [−0.248, −0.099] and β = −0.181, p = 0.002, 95% CI = [−0.262, −0.103], respectively), and from sports habits to resilience ( β = 0.152, p = 0.003, 95% CI = [0.061, 0.234]). Moreover, a strong effect of poor sleep quality on AL was found ( β = 0.127, p = 0.002, 95% CI = [0.05, 0.202]), and equivalent but opposed direct effects on poor sleep quality emerged from perceived influence ( β = 0.243, p = 0.001, 95% CI = [0.14,0.347]) and resilience ( β = −0.243 p = 0.003, 95% CI = [−0.325, −0.152]). A full mediation effect of the number of traumatic stressors on AL was found ( β = 0.025, p = 0.001, 95% CI = [0.01,0.05]) through the fourth path analysed ( ←Perceived Influence ←Sleep ←AL ). A partial mediation effect of sports habits on AL was also revealed through the fifth ( ←Resilience ←Sleep ←AL ) and sixth ( ←Resilience ←Perceived Influence ←Sleep ←AL ) evaluated pathways ( β = −0.005, p = 0.001, 95% CI = [−0.011,-0.002]; β = 0.000, p = 0.008, 95% CI = [−0.001, 0], respectively). For complete statistical details see Supplementary Table S10 . Section title: Sleep quality as pivotal mediator between life stressors and healthy habits on AL Educational score: 4.135604381561279 Domain: biomedical Document type: Study Language: en Similarly, when sleep was included in the path analysis for psychosocial stressors , significant direct effects on AL were found from the Pyramid score and sports habits ( β = −0.172, p = 0.002, 95% CI = [−0.242, −0.095]; β = −0.174, p = 0.003, 95% CI = [−0.253, −0.094], respectively), as also shown in the assessment of the first model. The direct effect of sports habits on resilience was also confirmed ( β = 0.148, p = 0.003, 95% CI = [0.049, 0.229]) and the partial mediation effect of sport on AL by perceived influence and resilience shown in the analysis of Model 1 now appear relayed via sleep quality through the fifth ( β = −0.004, p = 0.001, 95% CI = [−0.01, −0.002]) and sixth evaluated pathways ( β = 0.000, p < 0.001, 95% CI = [−0.001, 0]). Additionally, full mediation effects were found on AL through the perceived influence to sleep pathway from the number of psychosocial stressors ( β = 0.021, p = 0.001, 95% CI = [0.008, 0.04]) and from cognitive habits ( β = 0.001, p = 0.033, 95% CI = [0, 0.004]). Full statistical details are described in Supplementary Table S11 . Section title: Our findings Educational score: 3.6635336875915527 Domain: biomedical Document type: Study Language: en As previously reported, higher AL levels were shown in males, older ages and those with fewer years of education . Whereas higher engagement in sports was correlated to males, females correlated to higher Pyramid scores, and number and perceived influence of psychosocial stressors. Similar differences in healthy habits have been found previously in adolescents, midlife and older adult studies, with females more engaged in healthier diet and males in sports and physical activities . Additionally, higher psychosocial stress perception, emerged from family, job, finances or health issues, have been reported in females compared to males . Section title: Our findings Educational score: 3.3822691440582275 Domain: biomedical Document type: Study Language: en None of the healthy habits analysed or types of stressors showed correlations with age, whereas more education correlated with younger participants, engagement in cognitive habits, adherence to Mediterranean diet and, weaker but significantly to sport habits. These results agree with previous evidence showing that more educated people tend to adopt healthier lifestyle habits, particularly regarding diet and physical activity . Additionally, adherence to a Mediterranean diet was correlated with more frequent engagement in cognitive and sports activities, which could be explained by the significative relation between level of education and engagement in healthy lifestyles. Section title: Our findings Educational score: 2.317220449447632 Domain: biomedical Document type: Study Language: en Also, in line with previous studies, lower levels of AL were correlated with higher Pyramid diet scores , frequent engagement in sports and better sleep quality . Section title: Our findings Educational score: 1.880470633506775 Domain: biomedical Document type: Study Language: en Number of traumatic stressors were positively correlated with psychosocial events, as well as between their respective perceived influence. This may suggest that regardless of the type of stressor, the more events experienced in life, the greater distress will be perceived at present. Section title: Our findings Educational score: 2.2972443103790283 Domain: biomedical Document type: Study Language: en A higher number of both traumatic and psychosocial stressors, and their perceived influence, correlated with increased engagement in cognitive activities and poorer sleep quality. However, lower number and influence of psychosocial stressors correlated with increase sports practice, consistent with sex differences favouring males in these factors. Section title: Our findings Educational score: 2.58827543258667 Domain: biomedical Document type: Study Language: en Sleep quality showed no correlation with sex, age or years of education. However, greater engagement in sports was the only healthy habit correlated with better sleep quality and resilience abilities. Similar finding relating physical activity and sleep quality improvement have been extensively reported both in healthy children, adolescents and older adults , and people with impaired sleep such as insomnia or major depressive disorder . Section title: Our findings Educational score: 4.098962306976318 Domain: biomedical Document type: Study Language: en In our first analysis for traumatic stressor and AL mediated by resilience and perceived influence (Model 1), no effects of trauma were found, although significant direct effects on AL were shown from Pyramid diet scores and sport habits. The latter also exerted a significant direct effect on resilience. Similar results were found in the path analysis for psychosocial stressors through Model 1. Both results confirm previous findings associating sports and healthy diet with low AL levels and suggest that these habits could be preventing increased inflammatory, cardiovascular and metabolic factors, regardless of the influence from traumatic or psychosocial stressors. Interestingly, resilience showed a significant effect on the perceived influence of both psychosocial stressors and traumatic stressors, suggesting a potential impairment of resilience abilities after trauma as reported in PTSD . Moreover, engagement in sport habits also showed a positive association with resilience and exerted a significant indirect effect on AL through the relation between resilience and lower perceived influence of psychosocial stressors. Recent evidence reports similar enhancing effects on resilience by physical activity practice . Section title: Our findings Educational score: 4.155165672302246 Domain: biomedical Document type: Study Language: en Our second analysis of the effects of traumatic stressors on AL with sleep quality as additional mediator (Model 2) confirmed the significant associations between Pyramid score and sport habits on AL, and revealed a strong effect of sleep on AL, with poorer sleep quality associated with higher AL levels. Interestingly, opposed direct effects on poor sleep quality emerged from perceived influence and resilience, suggesting that the modulation of traumatic stress on AL might rely on a proportional balance between resilience ability and perceived influence over sleep quality. As previously reported, sleep is essential to adaptive processing of emotional experiences . It should therefore not be surprising to unveil an effect of traumatic stressors when sleep is included as additional mediator since a bidirectional relationship between impaired sleep and PTSD has been extensively studied , as well as the role it plays in promoting resilience and extinction of learned fear after traumatic events . Moreover, experimental evidence obtained through sleep deprivation protocols have shown that poor sleep quality can be considered as a stressor by itself that promotes high AL levels by increasing blood pressure, cortisol release, glucose resistance and inflammatory markers, among other effects . Section title: Our findings Educational score: 4.1690521240234375 Domain: biomedical Document type: Study Language: en The full mediation effect found between the number of traumatic stressors on AL through associations between perceived influence and sleep quality also agrees with studies on PTSD, where resilience ability is impaired to extinguish the perceived effects of traumatic stressors, In the absence of additional healthy habits modulators the amount of experienced traumatic events tips the balance towards increased levels of AL by outweighing the protective effect of resilience. Additionally, when sleep was added to the analysis of traumatic stressors, a partial mediation of sport habits on AL was revealed through a positive association to resilience, through both a direct effect of resilience on sleep and via perceived influence on sleep. Thus, the engagement in sports habits not only decreases AL by itself, but it can also potentiate the protective sleep-mediated effects of resilience on AL levels directly, and by tipping the balance against the effect of perceived traumatic stress on AL through poor sleep quality. Section title: Our findings Educational score: 4.071111679077148 Domain: biomedical Document type: Study Language: en When Model 2 was assessed for psychosocial stressor, the associations of diet and sport habits on AL, and the direct effect of sports on resilience shown by the first analysis were confirmed. A full mediation of the number of psychosocial stressors on AL was also unveiled through the association of perceived influence on sleep, in an equivalent magnitude to that seen for the case of the numbers of traumatic stressors, and in agreement with evidence reporting that sleep is affected by repeated stress regardless the kind of events experienced . Section title: Our findings Educational score: 4.079495429992676 Domain: biomedical Document type: Study Language: en Interestingly, a full mediation effect of cognitive habits on AL was found through positive association of perceived influence and sleep, suggesting that higher engagement in cognitive activities increases perceived influence of psychosocial stressors and worsen sleep quality. This result contradicts existing evidence associating lower AL with practice of cognitive lifestyle habits , and no studies have yet described a similar finding. However, such relation could be explained in part by the significant positive correlation found between engagement in cognitive activities and perceived influence of psychosocial stressors ( r = 0.159, p < 0.001). If we consider frequent involvement in cognitively stimulating activities as an indicator of higher intelligence, some studies have shown a positive association between that and self-awareness, worry, rumination and anxiety levels , suggesting that more intellectual individuals are more prone to develop rumination of negative experiences as an adaptive process to enhance problem solving , causing them frequent negative repetitive thoughts that may increase their experience of emotional distress . Section title: Our findings Educational score: 4.046720504760742 Domain: biomedical Document type: Study Language: en Finally, the partial mediation effect of sport habits on AL by perceived influence and resilience shown in the first analysis now appear relayed via sleep quality, in a similar way as shown in the assessment of traumatic stressors, through both a direct effect of resilience on sleep and via perceived influence on sleep. These results support the idea regarding the enhancement of resilience by physical activity and its protective on AL mediated by sleep, regardless of the type of stressor experienced. Altogether, the analyses of traumatic and psychosocial events through the second model revealed the pivotal role of sleep in the modulation of AL by healthy habits and life stressors. Section title: Conclusion and limitations Educational score: 4.181472301483154 Domain: biomedical Document type: Study Language: en As mentioned at the beginning of this work, high AL levels are caused by three possible situations: (i) a repeated stress exposure, reflected in our analyses by the number of life stressors; (ii) the inability to successfully adapt to stress demands through effective copying strategies, assessed through the resilience questionnaire; and (iii) the inability to terminate the physiological response to stress after the end of the event, also assessed through resilience levels and its relation to the amount of influence of the stressors perceived during the past year. Only by including sleep as mediator of the associations between life stressors and healthy habits on AL, the modulation by the three situations emerged: (i) the first through the effects of the number of events, whether traumatic or psychosocial; (ii) the second by the protective effect of resilience on AL by improving sleep quality; and (iii) the third, by increasing the weight of the association between perceived influence on poor sleep and surpassing the effect of resilience, in the case of traumatic stressors. This suggested a hypothetical proportional balance in the effects of perceived influence of stressors and resilience on sleep quality, such that the former acts to worsen it while the latter improves it . This balance mediates the modulation of AL levels by weighing the influence of stressors and healthy habits, tipping towards decreased AL when frequent engagement in sports practice is added to the equation . Section title: Conclusion and limitations Educational score: 4.007205963134766 Domain: biomedical Document type: Study Language: en However, a word of caution regarding these conclusions should be mentioned, as our results rely on a self-reported sleep questionnaire and not through objective sleep measurements. Previous studies have shown that self-reported perception of health and well-being could be negatively biased in stressed and highly deprived populations , pointing to the need to use more objective sleep measurements to further validate our analysis. As gold-standard polysomnography is unfeasible to be performed in large samples of participants, actigraphy measurements obtained through by wearable devices emerge as an option to consider in future assessments, not only because of their proved reliability but also due to the possibility to evaluate further sleep structures such as NREM and REM sleep amounts . | Review | biomedical | en | 0.999997 |
PMC11695330 | Section title: Introduction Educational score: 4.047093391418457 Domain: biomedical Document type: Review Language: en In the dynamic and often overwhelming pace of modern life, mental health has emerged as a global priority, with depression and anxiety being the most highly prevalent psychological problems. As some studies report, the prevalence of these emotional disorders is very high. The lifetime prevalence of depression symptomatology ranges between 2-21% ( 1 ) and near a 4% of the population suffers from anxiety disorders ( 2 ). This tendency has been increasing over the decades ( 2 , 3 ). These conditions are not merely individual problems but represent collective challenges that deeply affect our society in multiple dimensions, implicating socioeconomic consequences. At the individual level, these conditions can lead to a significant decrease in quality of life, interfere with the ability to maintain healthy personal and professional relationships, and in some cases, even lead to suicidal ideation and suicidal attempts ( 4 – 6 ). Further, mental health disorders are associated with economic losses due to lost productivity and healthcare costs ( 7 – 9 ). This context highlights the need for better understanding the mechanisms that can influence the appearance and maintenance of emotional problems, generating new models that permit to improve the prediction of their course and risk of relapses. Section title: Introduction Educational score: 3.9692163467407227 Domain: biomedical Document type: Study Language: en From a cognitive perspective, cognitive biases have been conceptualized as key mechanisms for the emergence of emotional symptomatology ( 10 – 14 ). From a traditional point of view, these biases are understood as tendencies to preferentially processing some types of emotional information over other ones, giving rise to differences in emotional and behavioral aspects as a consequence of this biased processing ( 14 ). One of the most studied biases is the interpretation bias, referred to the individual’s tendency to interpret ambiguous information in more negative or positive ways. For example, in a context or scenario where a boss does not greet his/her workers when he/she always use to do it, some individuals may generate a negative interpretation centered on interpreting that his/her boss is unhappy with their job, while others individuals may interpret this situation on a more neutral way, centered on the fact that his/her boss slept badly the last night. Several studies have assessed the presence of negative interpretation biases in different emotional problems, such as depression ( 15 ) and anxiety ( 16 ), showing the existence of differences between people with and without emotional symptoms, namely negative interpretation biases being higher in individuals with higher depression and anxiety levels ( 17 – 21 ). Consequently, negative interpretation biases seem to be relevant to understanding emotional dynamics, making it important to clarify their functioning, and factors involved in their occurrence. Section title: Introduction Educational score: 4.11353874206543 Domain: biomedical Document type: Review Language: en Following Beck’s traditional model [for later reviews, see ( 22 )], these biases would arise as a consequence of the interaction between a series of latent negative schemas in the individual and the occurrence of contextual stressors. Latent schemas refer to representations of stimuli, ideas or experiences that are internally stored in memory, giving rise to core internal beliefs that guide the interpretation of the experienced situations (e.g., “if I do not get to achieve everything that I have set out to achieve, I am a loser”). According to this framework, when a schema is triggered by a stressor, the meaning of the experienced situations is interpreted based on this schema, influencing all the subsequent cognitive, emotional, motivational, and behavioral processes. These latent negative schemas might have different contents. For example, the abovementioned Beck’s cognitive model proposed the existence of different nuclear schemas pertaining to beliefs about the world, the others, and oneself (i.e., self-referential schemas). In this sense, the activation of these self-referential schemas might influence the subsequent processes related to how an individual defines him/herself, which is understood as self-perception. Self-perception is, however, also defined by other specific self-domains. For example, the Self-Discrepancy Theory [SDT: ( 23 – 25 )], postulates the existence of different self-domains, namely, the actual-self (i.e., attributes that an individual truly believes they possess), ideal-self (i.e., attributes one ideally wants to possess) and the ought-self (i.e., attributes one believes they should possess). It is the discrepancies among these self-domains that have consistently been associated to different emotional problems. Concisely the SDT pinpoints actual versus ideal-self (i.e., actual-ideal) discrepancies to be related to higher levels of depressive symptoms and actual versus ought-self (i.e., actual-ought) discrepancies to be related to higher levels of anxiety symptoms ( 23 – 26 ). Section title: Introduction Educational score: 4.056848526000977 Domain: biomedical Document type: Study Language: en Thus, it is the discrepancies between the different self-domains that offer a closer look at the processes related to self-perception. Given that self-referential schemas cannot be directly measured, assessing indices of self-discrepancies could offer an indirect evaluation of Beck’s former schemas. In fact, this idea has been considered not only in theoretical models [e.g., ( 23 – 26 )], but also in experimental studies [e.g., ( 27 )]. For example, some studies have shown the activation of certain latent schemas through priming effects of different self-discrepancies ( 27 ). These studies seek to establish the differentiating role of specific self-discrepancies in the emergence of specific emotions, in order to establish whether their activation can affect the emotional state of the individual. For instance, in the study of Strauman and Higgins ( 27 ), participants were differentiated according to their levels of self-discrepancies (i.e., high actual-ideal self-discrepancies and high actual-ought self-discrepancies). Participants were exposed to priming based on 3 different conditions (i.e., relevant but not discrepant attributes, relevant and discrepant attributes and a yoked condition). Results showed that only those participants exposed to a discrepancy-relevant priming condition showed a change in their levels of certain emotions (i.e., dejected or agitation) depending on the type of predominantly primed discrepancy and the type of relevant self-discrepancy of each participant. Therefore, according to this type of former evidence, discrepancies between the actual-self and other domains could serve as indicators of predominantly active latent cognitive schemas, which, when interacting with contextual stressors, could facilitate an increase in the salience of such mismatch, thus generating the characteristic emotional responses to each type of self-discrepancy ( 27 ). Consequently, the way in which the individual defines him/herself and, above all, the degree of discrepancy with their other self-domains (i.e., ideal or ought) seem to be a variable of relevance to study the emergence of emotional symptomatology, and their potentially intervening mechanisms (i.e., resulting negative interpretation biases). Section title: Introduction Educational score: 4.130515098571777 Domain: biomedical Document type: Study Language: en Consequently, in this study, we aimed to test the explanatory mechanisms of emotional symptoms from traditional cognitive models (e.g., existence of a latent cognitive scheme that would produce biases in the processing of information when interacting with a stressor) through the evaluation of these schemes in relation to the existence of self-discrepancies. The integration of these frameworks could help to improve the understanding of causal factors for the occurrence of emotional symptoms. Yet, the relationships between self-discrepancies and interpretation biases and their specific paths of influence in changes in emotional symptoms have not been previously tested yet. Thus, we tested mediational models through which self-discrepancies would act as latent self-referential cognitive schemas, which would facilitate higher levels of negative self-referential interpretation biases and, consequently, explaining the presence and change of emotional symptoms. Specifically, we analyzed the relationships between individual differences in self-discrepancies (i.e., actual-ideal, ought-ideal), negative interpretation biases, and emotional symptoms (i.e., depression, anxiety), considering the mediating role of negative interpretation biases in the relationship between self-discrepancies and emotional symptoms both cross-sectionally and longitudinally. To do this, undergraduate participants were asked to perform two tasks, one assessing self-discrepancies [i.e., actual-ideal self-discrepancies and actual-ideal self-discrepancies; ( 28 ) and another one assessing negative interpretation biases ( 29 )]. In order for participants to respond based on how they perceived themselves, both tasks used clearly self-referential stimuli. Similarly, symptoms of depression and anxiety were assessed after completing the tasks and approximately one-two month later, right before starting undergraduates’ exam period, which previous research has identified as a naturalistic major stressor for the undergraduate population under study ( 30 ). This methodology allowed us to test individual differences in the change in symptoms when confronting a specific stress situation . Overall, it was hypothesized that higher levels of self-discrepancies would have a significant and positive direct relationship with higher negative interpretation biases, such that the greater the magnitude of the self-discrepancy, the higher the level of the bias. Furthermore, it was hypothesized that higher negative interpretation biases would have a mediating role in the relationship between higher self-discrepancies and higher emotional symptom levels. Section title: Research type and design Educational score: 4.034412860870361 Domain: biomedical Document type: Study Language: en This study employs a quantitative, observational, non-experimental approach with a mediational and longitudinal design. This design was used to examine the role of negative interpretation biases as a mediator in the relationships between self-discrepancies (actual-ideal and actual-ought) and emotional symptoms (depression and anxiety). The cross-sectional analysis was conducted to test the relationships between variables at a single time point, while the longitudinal analysis tested these relationships when assessing changes in emotional symptoms over time, specifically in response to a natural stressor (exam period). Section title: Ethical aspects Educational score: 1.6572431325912476 Domain: biomedical Document type: Study Language: en The study was conducted in accordance with the ethical guidelines of the Declaration of Helsinki. Approval was obtained from the university’s ethics committee . Participants were informed about the objectives of the research and provided written informed consent before participating. They were assured of the confidentiality and anonymity of their data, as well as their right to withdraw from the study at any time without any consequences. Additionally, all participants received course credits as compensation for their involvement in the study. Section title: Population, sample and sampling Educational score: 2.616828680038452 Domain: biomedical Document type: Study Language: en The sample was composed by 73 (61 females, 12 males) university students from Complutense University of Madrid with a mean age of 21.45 years (SD = 3.57). Of the total sample, 72 participants (60 females; mean age = 21.47; SD age = 3.59) completed a follow-up in which their levels of emotional symptoms were assessed again just before the beginning of the exam period, an event that has been identified as a relevant stressor for university students ( 30 ). The inclusion criteria for the study were as follows: (1) undergraduate students enrolled at Complutense University of Madrid, (2) native Spanish speakers, and (3) individuals with normal or corrected-to-normal vision. Recruitment was conducted by advertising the study in various classes, offering university credits as compensation. Section title: Depressive symptoms Educational score: 3.960421323776245 Domain: biomedical Document type: Study Language: en The Patient Health Questionnaire (PHQ-9) ( 31 , 32 ) is a nine-item questionnaire that evaluates depressive symptoms’ severity during the previous two weeks in a four-point scale (from 0 to 27). Higher values indicate higher levels of depressive symptoms. In this study, the PHQ-9 showed a high internal consistency at both the baseline (α = .84) and the follow up (α = .88). Section title: Anxiety symptoms Educational score: 3.7919321060180664 Domain: biomedical Document type: Study Language: en The Generalized Anxiety Disorder Scale (GAD-7) ( 33 , 34 ) is a seven-item questionnaire that measure the severity of anxiety symptoms in the previous two weeks in a four-point scale (from 0 to 21). Higher values indicate higher severity of anxiety symptoms. In this study, the GAD-7 showed a high internal consistency at both the baseline (α = .89) and the follow-up (α = .89). Section title: Self-discrepancies Educational score: 3.7569143772125244 Domain: biomedical Document type: Study Language: en In order to evaluate the different types of self-discrepancies based on Higgins’ model ( 23 – 25 ), the SCQ-CC [Self-Concept Questionnaire-Conventional Construct: ( 28 )] was used. This task allows evaluating the different self-domains (i.e., actual, ideal and ought) to calculate the differences between them, thus generating self-discrepancy indices [i.e., actual-ideal self-discrepancy and actual-ought self-discrepancy; see ( 23 – 25 , 27 )]. To do this, participants had to rate 28 self-referent adjectives (e.g., cheerful, worrying, organized, warm), on a scale from 1 to 7, to what extent those attributes described them for each of the different types of self-domains defined by Higgins’ theory (i.e., actual, ideal and ought), forming a total of 84 items. For this study, the task thus focused on assessing the participant’s own perception of him/herself, not including other standpoints such as the participant’s idea of how other relevant people think about him/her (i.e., standpoint ‘others’). Therefore, the discrepancy indices calculated were referred to the individual’s perspective on himself (actual own-ideal own self-discrepancy and actual own-ought own self-discrepancy) ( 23 – 25 ). Section title: Self-discrepancies Educational score: 3.6299397945404053 Domain: biomedical Document type: Study Language: en Self-discrepancies were calculated following the same method as in previous studies [e.g., ( 28 )]. In this case, actual-ideal self-discrepancy was calculated averaging the absolute difference between the scores of the self-reports from actual and ideal self. The actual-ought discrepancy was calculated in the same way, but only referring to the absolute differences between the scores of the self-reports from actual and ought self. Thus, two self-discrepancy indices were computed for each participant, actual-ideal and actual-ought, that ranged from 0 (no discrepancy) to 6 (maximum discrepancy). In this task, the self-discrepancies index showed good internal consistencies, for both actual-ideal (α = .76) and actual-ought (α = .80). Section title: Interpretation bias Educational score: 4.057931900024414 Domain: biomedical Document type: Study Language: en To assess the degree of negative interpretation biases, a computerized version of the Scramble Sentence Task (SST) ( 29 ) was used. In this task, participants must form grammatically correct sentences with 5 out of the 6 words that appear scrambled on the screen, and that can be solved into positive or negative unscrambled sentences, thus assessing the participants’ tendency to solve ambiguous material in specific emotional ways. This task has demonstrated its validity to evaluate negative interpretation bias in other studies with similar methodology ( 21 , 35 – 37 ), and shown capacity to differentiate the degree of biases at different stages of emotional problems (i.e., dysphoric, clinically depressed, formerly depressed) ( 21 ). The task for this study comprised 30 items. All trials started with a fixation cross on the left side of the screen during 1500 ms, which the participants were asked to look at. Next, the reading phase started , where participants had to read from left-to-right the 6 words that appeared for 8000 ms, in order to mentally unscramble the material to form a grammatically correct sentence using only 5 out of those words. After this time, the response phase began and the participants, using the mouse, had to click over the different boxes where the words appeared in order to form the previously unscrambled sentence. To do this, they had a limited time of 9000 ms. After that time limit, if a full response had not been indicated, the task moved to the next trial. Previous to complete the actual task, participants had to complete 5 practice items based on unscrambling neutral sentences (e.g., ‘I really like eating grapes/bananas’), to get familiar with the procedure, before completing the 30 emotional items of the main task. Section title: Interpretation bias Educational score: 3.31485915184021 Domain: biomedical Document type: Study Language: en The emotional sentences of the task (e.g., born winner a am loser I) could be solved into a positive (e.g., I am a born winner) or a negative (e.g., I am a born loser) solution. Thus, the number of negatively unscrambled sentences was divided by the total number of unscrambled sentences to index the tendency of participants to interpret ambiguity in a negative manner. To avoid a bias due to the position of the emotional words, it was controlled that negative and positive words appeared the same proportion of times in left- and right-positions of the screen (second and fifth boxes) across the trials. The negative interpretation bias index ranged from 0 to 1, with higher values indicating a higher negative interpretation bias. To obtain an accurate index, only those participants who solved at least 20 sentences out of the 30 sentences correctly were included. Using this criterion, no participants had to be eliminated since all of them completed at least 20 trials correctly. Section title: Procedure Educational score: 3.3293795585632324 Domain: biomedical Document type: Study Language: en Upon arriving at the laboratory, participants were informed about the purpose of the study and were asked to sign the informed consent form if they agreed to participate. They were then instructed to start completing the paper-and-pencil version of the SQC-CC ( 28 ), followed by the SST ( 29 ) via e-Prime 3.0, and finally to complete the online questionnaires assessing depression and anxiety via Qualtrics. The experimental session lasted less than 60 minutes on average. Approximately one-two months later, just before the exam period, participants were recontacted and asked to complete the same set of questionnaires as during the experimental session to obtain the longitudinal measures of symptoms’ change. Section title: Data-analysis plan Educational score: 4.104427337646484 Domain: biomedical Document type: Study Language: en To assess the relationships between self-discrepancies, interpretation biases, and emotional symptoms, a series of steps were taken based on the established hypotheses. First, correlations between the study’s relevant variables (self-discrepancies, interpretation biases, and emotional symptoms) were calculated using SPSS version 27.0 to evaluate the initial relationships between them. Next, mediation models were constructed with the key variables at T1, where self-discrepancies served as the independent variables, negative interpretation bias as the mediator, and emotional symptoms (depression and anxiety) as the dependent variables. Standardized indices were calculated to facilitate the interpretation of the different effects tested in the models (i.e., direct and indirect effects). Further, the same models were tested longitudinally (i.e., considering changes in emotional symptoms from T1 to T2, at the confrontation of the stressor). To do this, standardized residuals of emotional symptoms at T1 predicting themselves at T2 were computed, as indices of change in depression and anxiety at the face of stress. These indices of change were then entered as the dependent variables in the longitudinal models, allowing to test how self-discrepancies at T1 predicted emotional symptoms’ changes, directly and indirectly through the mediation of negative interpretation biases. Standardized residuals are a widely used method for assessing change beyond simple differences between time points ( 38 , 39 ). Specifically, this parameter is obtained by calculating the residuals resulting from performing a linear regression where the symptoms (whether depressive or anxious) at T1 predict the symptoms (whether depressive or anxious) at T2. Thus, the obtained standardized residual values represent the proportion of variance in symptoms’ change not explained by the previous levels of symptomatology (i.e., T1 scores) 1 . All mediation models were computed using JASP version 18.2. Section title: Results Educational score: 4.047556400299072 Domain: biomedical Document type: Study Language: en Bivariate correlations between the variables of the study were first computed. Results indicated that both types of self-discrepancies were related with higher levels of depressive ( r a c t − i d e S D = .51 , p < .001 ; r a c t − o u g S D = .53 , p < .001 ), anxiety symptoms ( r a c t − i d e S D = .56 , p < .001 ; r a c t − o u g S D = .52 , p < .001 ) and with higher levels of negative interpretation biases ( r a c t − i d e S D = .56 , p < .001 ; r a c t − o u g S D = .54 , p < .001 ) at T1. In the same way, higher levels of negative interpretation biases were related with higher levels of depressive ( r = .65, p <.001) and anxiety ( r = .56, p <.001) symptoms. Both types of self-discrepancies were highly related ( r = .74, p <.001). By last, no discrepancy indices nor negative interpretation bias were related to symptom change indices from T1 to T2 (all r’s<.14 & > -.07). In Supplementary Table 1 appears the mean and standard deviation and in Supplementary Table 2 the specific correlation indices of variables implicated in the study. Section title: Cross-sectional models with actual-ideal self-discrepancies as predictor Educational score: 3.8502249717712402 Domain: biomedical Document type: Study Language: en After initially verifying a relationship between the study variables, it was decided to carry out mediational models with each of the self-discrepancy variables as predictors, negative interpretation bias as the mediator and emotional symptoms’ indices as outcome measures. Following ( 17 , 35 , 37 , 40 ), mediational effects were tested through bootstrapping procedures. In this case, 95% confidence intervals with 5000 bias-corrected bootstrap samples were estimated, thus an indirect effect was significant if the confidence interval did not include 0. Section title: Cross-sectional models with actual-ideal self-discrepancies as predictor Educational score: 3.9726710319519043 Domain: biomedical Document type: Study Language: en As for the models considering the actual-ideal self-discrepancy as the predictor, its direct effect was positive and significant for both types of symptoms: depressive (β:.51, SE :.25, z = 2.05, p = .04) and anxious symptom levels (β:.86, SE :.26, z = 3.3, p <.001). Likewise, a higher actual-ideal self-discrepancy was also significantly related to higher levels of negative interpretation bias (β:1.33, SE :.23, z = 5.72, p <.001). Finally, a higher negative interpretation bias was related to higher levels of both depressive (β:.53, SE :.1, z = 5.003, p <.001) and anxiety symptoms (β:.36, SE :.11, z = 3.29, p = .001). Figure 4 shows the different direct relationships between the study variables. Section title: Cross-sectional models with actual-ideal self-discrepancies as predictor Educational score: 4.085845470428467 Domain: biomedical Document type: Study Language: en Regarding the indirect effects, the analyses supported a significant indirect effect of negative interpretation bias in the relationship between the actual-ideal self-discrepancy and depression ( β :.7, SE :.19, z = 3.77, p <.001, CI:.37, 1.16) and anxiety symptom levels ( β :.48, SE :.17, z = 2.85, p = .004, CI:.18,.95), thus supporting that that actual-ideal self-discrepancies were related to both forms of symptomatology indirectly through their relation with a higher activation of negative interpretation biases. Section title: Cross-sectional models with actual-ought self-discrepancies as predictor Educational score: 3.71650767326355 Domain: biomedical Document type: Study Language: en As for the actual-ought self-discrepancies, the results showed a similar pattern. In this case, actual-ought self-discrepancies were positive and directly related to both depressive ( β :.51, SE :.21, z = 2.5, p = .01) an anxiety symptom levels ( β :.6, SE :.22, z = 2.72, p = .006), as well as with negative interpretation biases ( β : 1.09, SE :.2, z = 5.44, p<.001). In the same way, negative interpretation biases were highly associated with both depressive ( β :.51, SE :.1, z = 4.98, p <.001) and anxiety ( β :.4, SE :.11, z = 3.63, p <.001) symptoms . Section title: Cross-sectional models with actual-ought self-discrepancies as predictor Educational score: 4.023885726928711 Domain: biomedical Document type: Study Language: en Regarding the indirect effects of these models, the results supported significant indirect effects of negative interpretation bias in the relationship between actual-ought self-discrepancies and both forms of emotional symptoms, depression ( β :.55, SE :.15, z = 3.67, p <.001, CI :.32,.87) and anxiety ( β :.43, SE :.14, z = 3.02, p = .003, CI :.17,.82). Therefore, although there was a direct effect of the level of actual-ought self-discrepancies on both forms of symptomatology, part of this effect occurred indirectly through their influence on a higher activation of negative interpretation biases. Section title: Longitudinal models with actual-ideal self-discrepancies as predictor Educational score: 4.0937886238098145 Domain: biomedical Document type: Study Language: en Focusing on the longitudinal model (i.e., testing the mediation role of negative interpretation bias in the relation between actual-ideal self-discrepancies and the change in emotional symptoms from T1 to T2, indexed through standardized residuals), the results showed differences with respect to the ones from the cross-sectional models. In this case, results showed that higher actual-ideal self-discrepancies at T1 were not directly related to changes in emotional symptoms for both depression ( β : -.13, SE :.34, z = -.38, p = .71; and anxiety symptom ( β : -.53, SE :.33, z = -1.6, p = .11), while their association with higher levels of negative interpretation bias remained significant ( β : 1.33, SE :.23, z = 5.72, p <.001). Negative interpretation bias did not predict changes in depressive symptoms ( β : -.02, SE :.14, z = -.13, p = .89), although there was a trend to predict increases in anxiety symptoms ( β :.26, SE :.14, z = 1.91, p = .06) . Section title: Longitudinal models with actual-ideal self-discrepancies as predictor Educational score: 4.056514739990234 Domain: biomedical Document type: Study Language: en Regarding the indirect effects, the results supported a mediational effect of higher actual-ideal self-discrepancies predicting increases in anxiety symptoms through their relationship with a higher activation of negative interpretation biases ( β :.35, SE :.19, z = 1.81, p = .07, CI :.03,.71). In contrast, this mediational effect was not supported for the model considering changes in depression symptom levels ( β : -.02, SE :.19, z = -.13, p = .89, CI : -.37,.32). Section title: Longitudinal models with actual-ought self-discrepancies predictor Educational score: 3.9187631607055664 Domain: biomedical Document type: Study Language: en Finally, longitudinal models were conducted considering actual-ought self-discrepancies as the predictor (i.e., testing the mediation role of negative interpretation bias in the relation between actual-ought self-discrepancies and the change in emotional symptoms measured through standardized residuals). The direct effect of actual-ought self-discrepancies on the change in emotional symptomatology, either depressive ( β : -.03, SE :.28, z = -.1, p = .92) or anxious symptom levels ( β : -.27, SE :.28, z = -.98, p = .33) were both not significant. The association between actual-ought self-discrepancies and negative interpretation bias remained significant ( β : 1.09, SE :.2, z = 5.44, p <.001). Negative interpretation biases did not predict the change in emotional symptoms, for both depression ( β : -.04, SE :.14, z = -.3, p = .76) and anxiety ( β :.21, SE :.14, z = 1.55, p = .12) . Section title: Longitudinal models with actual-ought self-discrepancies predictor Educational score: 3.7220466136932373 Domain: biomedical Document type: Study Language: en As for indirect effects, they were both non-significant, indicating that actual-ought self-discrepancies did neither predict changes in emotional symptoms through their relationship with negative interpretation bias ( β : -.04, SE :.15, z = -.3, p = .76, CI : -.27,.22; β :.23, SE :.15, z = 1.49, p = .14, CI : -.06,.59, for depression and anxiety, respectively). Section title: Discussion Educational score: 3.8839738368988037 Domain: biomedical Document type: Study Language: en The way we define ourselves, especially the differences between how we actually perceive ourselves in comparison to other self-domains (e.g., ideal or ought), seems to be a key variable in understanding the experience of multiple emotional responses. Self-discrepancies can be understood as latent self-referential cognitive schemas, increasing certain cognitive biases in emotional information processing. In turn, a higher degree of these biases can facilitate more maladaptive emotional dynamics ( 23 , 25 – 27 ). However, no previous studies have integrated the study of all these processes together. In consequence, in this study we aimed to analyze the relationships between these variables and test the capacity of self-discrepancies to activate specific information processing biases (i.e., negative interpretation biases), and whether this would result in higher levels of emotional symptoms. Overall, the results support the mediating role of negative interpretation bias in the relationship between higher self-discrepancies and higher emotional symptom levels. Greater self-discrepancies were related to a pattern of more biased negative information processing, which in turn was related to higher levels of emotional symptoms. This mediation was fully supported for both types of self-discrepancies in cross-sectional models and was partially supported in longitudinal models. The indirect effect of negative interpretation bias was specifically supported only for the role of actual-ideal self-discrepancies in predicting changes in anxiety symptoms. These results are discussed below. Section title: Support of Higgins’ model Educational score: 4.1306376457214355 Domain: biomedical Document type: Study Language: en First, in this study we aimed to analyze the support of predictions from Higgins’ framework on the role of different self-discrepancies on different forms of emotional symptomatology ( 23 – 25 ). The results indicated that, contrary to the specificity proposed by this model (i.e., higher actual-ideal self-discrepancy being related to higher depressive symptoms and higher actual-ought self-discrepancy being related to higher anxiety symptoms), both self-discrepancy indices were related to higher levels of both types of emotional symptoms. These relationships were supported by correlational and cross-sectional mediation analyses. This inconsistency with the model’s prediction of specificity has been observed in other studies, where various combinations of relationships between self-discrepancies and emotion symptoms have been found. For example, some studies support the association of actual-ideal discrepancy with both types of symptoms ( 41 , 42 ), while other studies support the associations of actual-ought self-discrepancy with only depressive symptoms ( 43 ) or both emotional symptoms ( 44 ). Further, other previous studies have not found differences in actual-ideal and actual-ought self-discrepancies between clinically depressed and anxious participants ( 45 ). This contrast with the results of other studies that fully support Higgins’ model assumptions on specificity ( 23 – 25 , 46 , 47 ). These apparent inconsistency between results can be influenced by the type of task used in each study. Some studies have relied in idiographic tasks (i.e., assessing self-discrepancies through the election of unique personal traits/adjectives that define their understanding of themselves) whereas others make use of nomothetic tasks (i.e., assessing self-discrepancies based on a preestablish list of traits/adjectives). Some authors support the former ( 46 ), while others claim a high correlation between the indices, arguing the use of both types of tasks ( 28 , 48 ). Consequently, the results might suggest the existence of a transdiagnostic variable rather than multiple subindexes associated with specific symptomatic patterns ( 49 ). Section title: Support of the relation between self-discrepancy indices and interpretation biases Educational score: 4.111847400665283 Domain: biomedical Document type: Study Language: en As part of the central hypotheses of the study, the effect of self-discrepancies on promoting specific negative information processing biases were tested. The results support this assumption, showing a high degree of association between both types of self-discrepancies and negative interpretation biases across all types of analysis. None of the specific discrepancy indices exhibited a greater or lesser relationship with negative interpretation bias levels. In future studies, different types of negative interpretation bias (i.e., regarding solving ambiguity for specifically relevant depression and anxiety topics) could provide additional information regarding the specificity on relationships between self-discrepancies and negative interpretation biases. This relationship between self-referential discrepancies and other forms of negative interpretation biases has been observed in studies on eating disorders, where patients exhibited a negative bias in their body interpretation. In fact, this interpretation bias was correlated with body dissatisfaction, a key aspect in defining identity ( 50 ). Thus, these results suggest the potential importance of self-referential discrepancies ( 23 – 25 ) to explain the emergence and maintenance of emotional symptoms, as they may reflect specific activation of negative cognitive schemas ( 22 ). Following that logic, our results (i.e., through correlational and cross-sectional mediational analysis) can be seen as a further support for predictions from traditional cognitive models, which state that cognitive schemas produce changes in information processing patterns through cognitive biases, leading to characteristic emotional symptoms ( 22 ). Section title: Support of the mediational role of interpretation biases in the relationship between self-discrepancies and emotional symptoms Educational score: 4.127139091491699 Domain: biomedical Document type: Study Language: en Lastly, the mediating role of interpretation bias in the relationship between self-discrepancies and emotional symptoms was tested. The results support the predictive value of higher negative interpretation bias on higher levels of both types of emotional symptoms cross-sectionally, yet only for changes in anxiety in the longitudinal mediational models. Cross-sectional results are supported by other studies that have found direct associations between negative interpretation bias and both forms of emotional symptoms [e.g., ( 15 , 17 , 19 , 20 )]. However, the longitudinal value of cognitive biases to predict both forms of symptoms has not been deeply explored in previous literature, with some studies supporting its predictive value for both forms of symptoms ( 20 , 51 , 52 ), while others do not reach that assumption so clearly ( 53 ). These results could be due to several differences among studies, such as the type of sample investigated (i.e., clinical vs. subclinical), the lack of consideration of emotion regulation strategies or contextual factors influencing data collection. Regarding this, multiple models propose that the effect of biased information processing to predict changes in symptoms would depend on how biases influence subsequent emotional regulation processes, rather than directly influencing on the symptoms (e.g., 12 ). Another reason for the only prediction on anxiety change could be due to the contextual component of the study, as the longitudinal data collection was designed to detect symptom changes in response to an upcoming stressful event (the exam period). For this reason, it is possible that the time at which the data were collected may had particularly exacerbated changes in anxiety when facing an imminent stressful situation (e.g., exam period) as a common mechanism when trying to face an event that is interpreted as challenging. Following this logic, a better detection of individual differences in changes in depressive symptoms as a result of this particular stressor, would require to further assess emotional symptoms at the end of the event (i.e., after having completed the exam period and getting the final grades). This could at least partly explain a higher room to detect changes in anxiety and the mechanisms implicated in predicting that change in the current study, contrasting with the null results found for the longitudinal mediational models considering depression change as an outcome. Section title: Support of the mediational role of interpretation biases in the relationship between self-discrepancies and emotional symptoms Educational score: 4.09439754486084 Domain: biomedical Document type: Study Language: en Overall, whereas there are previous studies evaluating the role of discrepancies as cognitive schemas on emotional symptoms, none to date have integrated the study of their influence on negative information processing and the various mediating effects of the latter on the relationships between self-discrepancies and emotional symptoms. Moreover, conducting not only cross-sectional but also longitudinal models allowed us to test the predictive value of discrepancies and biases in symptom change. The study thus holds significant clinical value by providing initial insights into how identity-related variables can influence the processing of ambiguous information, thereby potentially triggering emotional symptoms in the face of a stressor. From a clinical perspective, there are multiple implications associated with the study. Firstly, although longitudinal models did not show clear results, different mechanisms through which intervention could be designed to reduce emotional symptoms were observed. Both self- discrepancies (associated with a self-referential component) and negative interpretation biases seem to be linked to each other, so changes in one could cause changes in the other. Therefore, the management or resolution of self-discrepancies (e.g., cognitive behavioral therapy) could facilitate the reduction of negative biases. Similarly, interventions designed to modify these cognitive biases ( 54 ) has shown how such reduction can relate to improvements in emotional symptoms which could in turn influence the future activation of negative cognitive schemes. Section title: Limitations Educational score: 4.117523670196533 Domain: biomedical Document type: Study Language: en Despite the relevance of these findings, several limitations must be considered. Firstly, the sample consisted predominantly of undergraduate students, mostly female. This limits the representativeness of the findings and reduces the generalizability to broader social groups (e.g., individuals of different ages, occupations, and stress levels). In consequence, results may reflect the experiences of a specific social group rather than those ones of the general population, due to both the sample size and its characteristics. Secondly, the study focuses on an educational context, particularly the exam period as a natural stressor. This situation may be too specific to students and may not generalize to other social groups or life contexts. This limits the extrapolation of results to other types of stressors (e.g., workplace or family-related stress), which could activate different cognitive schemas and yield different interpretation biases. These limitations warrant further replication in broader samples and considering different forms of stress context in future research. Thirdly, other limitations arise from the types of statistical analyses used in the study. The accuracy of mediational models is sensitive to sample size, which in this study may lead to findings that are specific to the sample rather than reflective of a broader population. With a small sample size, the power to detect subtle effects might be reduced, and the chance of sample-specific results increased. Although bootstrapping was employed to enhance the robustness of results’ estimation, a larger sample would provide a more reliable basis for testing specific mediational relationships among these factors in future studies. Finally, the timing of longitudinal data collection may have influenced results, as the assessment of emotional symptom changes occurred just before the stressor onset. This timing might have contributed to the detection of changes primarily in anxiety rather than depression, potentially limiting the assessment of the predictive roles of self-discrepancies and negative interpretation biases in depression specifically. Section title: Limitations Educational score: 4.067617416381836 Domain: biomedical Document type: Study Language: en As such, our results should be interpreted with caution. Future studies should increase sample size and diversify the sample to include different social characteristics (e.g., a more balanced gender ratio and a broader age range), providing a more robust pattern of results. Additionally, future research should consider testing the relationships between self-discrepancies and negative interpretation biases in clinical populations. Such studies could yield valuable insights into how cognitive schemas and biases may contribute to the maintenance or recurrence of emotional symptoms following specific stressors. Further, adding a follow-up assessment of emotional symptoms at the stressor’s conclusion (e.g., after the exam period and receipt of final grades) could capture a wider range of symptom variability, allowing distinctions between individuals who recover from the stressor and those whose symptoms persist or intensify. This expanded time frame would provide a more precise temporal context for analyzing how self-referential mechanisms predict emotional symptom dynamics over time, offering a clearer perspective on symptom change patterns. Section title: Conclusions Educational score: 3.8122150897979736 Domain: biomedical Document type: Study Language: en In conclusion, the results of this study show the importance of self-discrepancies as self-referential mechanisms that are relevant in explaining emotional symptoms. Results support the idea that self-discrepancies could be conceptualized as facets of the activation of latent cognitive schemas that would in turn facilitate the activation of negative biases during the processing emotional information, lastly influencing the appearance and/or maintenance of different emotional symptoms. | Review | biomedical | en | 0.999997 |
PMC11695336 | Section title: Introduction Educational score: 3.7711598873138428 Domain: biomedical Document type: Review Language: en Urinary incontinence (UI) is a common disorder in the population, usually increasing in prevalence with age ( 1 ). The unintentional loss of pee during urine storage is classified as urinary incontinence ( 2 ). Senior age, High BMI, menopause, and childbearing have all been linked to an increased risk of UI, according to previous research ( 3–6 ). Stress UI (SUI), Urge UI (UUI), and mixed UI (MUI) are the three most prevalent forms of UI ( 7 ). The ageing of the world’s population is making UI a serious public health issue. Section title: Introduction Educational score: 3.8547933101654053 Domain: biomedical Document type: Review Language: en The sudden, overwhelming urge to urinate followed right away by involuntary incontinence is known as urge urinary incontinence (UUI) ( 8 ). According to estimates, UUI affects a significant percentage of people in the US population—2.6 to 20.9% of males and 9.3 to 30.8% of women—and its incidence rises sharply with age ( 9 , 10 ). UUI can be a debilitating illness that causes significant deficits in self-confidence and psychological well-being in addition to a reduction in social contacts and interpersonal connections ( 11 ). These symptoms have a substantial negative influence on the quality of life and frequently call for behavioural, pharmaceutical, or surgical therapies. Section title: Introduction Educational score: 4.062612533569336 Domain: biomedical Document type: Study Language: en Diet as a part of behavioural therapy is now being considered a promising treatment for many diseases ( 12–15 ). The composite dietary antioxidant index (CDAI) is based on a range of dietary vitamins and minerals with antioxidant properties, including carotenoids, zinc, selenium, and vitamins C, E, and A. This summary score is used to assess a person’s dietary total antioxidant capacity (TAC) ( 16 , 17 ). Recent research indicates a link between CDAI and inflammatory biomarkers ( 18 ). It is widely recognized by scholars that considering a composite diet is more representative of actual daily nutrient intake in humans, rather than focusing on individual nutrients ( 19 ). Therefore, this study explores the relationship between the combination of the six trace nutrients mentioned above and UUI. Section title: Introduction Educational score: 3.9485085010528564 Domain: biomedical Document type: Study Language: en Numerous investigations have now demonstrated the connection between illnesses and CDAI ( 20–24 ). It’s still unclear, though, how CDAI and UUI are related. Previous studies have focused on the relationship between inflammation-related indices and female UUI, but there is relatively little research on inflammation and UUI in men. The National Health and Nutrition Examination Survey (NHANES) 2011–2018 data are used in this cross-sectional study to investigate any possible relationship between CDAI (and its components) and UUI prevalence in men. We also examined the potential pathways, and our findings should serve as a foundation for an exogenous antioxidant diet that prevents UUI. Section title: Information sources consulted for this study Educational score: 2.4113049507141113 Domain: biomedical Document type: Study Language: en One of the most important programs of the National Center for Health Statistics (NCHS) is the NHANES. Data that is indicative of the nation on the general health of the US population has been collected ( 25 ). With the use of a sophisticated multistage probability methodology, this program produces a nationally representative sample of non-institutionalized Americans ( 26 ). Since 1999, it has used a sophisticated, stratified, multi-stage probability sampling method to gather data from about five thousand people each year ( 27 ). IN addition, its survey cycle lasts 2 years. All procedures were approved by the NCHS Research Ethics Committee, and each participant gave their informed permission ( 28 ). Data from questionnaires, laboratory test indicators, physical examination characteristics, and sociodemographic status were also collected. There are extensive details about the program on the website. 1 Section title: Study population in this investigation Educational score: 3.927764654159546 Domain: biomedical Document type: Study Language: en This study analysed data from four NHANES cycles, spanning from 2011 to 2018. The survey was completed by 39,156 people in total (Representing an actual population of 103,877,855). Firstly, we excluded females ( n = 19,848) and males under 20 years of age ( n = 8,361). The subsequent exclusion criteria were as follows: (1) information about dietary ( n = 2,815) is unknown; (2) interviewees who had not finished the UI survey ( n = 304); (3) data about education status ( n = 7) is unknown; (4) body mass index ( n = 72) is unknown; (5) data about smoking status ( n = 3) is unknown; (6) information about vigorous activities ( n = 3) is unknown; (7) information about moderate activities ( n = 5) is unknown; and (8) Total cholesterol ( n = 3) is unknown. Finally, the study comprised 7,735 individuals in total after screening procedures, including 1,247 men with UUI and the remaining without UUI. Figure 1 shows the screening procedures. Section title: Measurement of CDAI Educational score: 2.9375975131988525 Domain: biomedical Document type: Study Language: en The 24 h dietary recall interview is the current section of the NHANES nutritional evaluation. Dietary interviewers with training who are fluent in both Spanish and English perform dietary recall interviews in person. Through discontinuous two-day, 24 h dietary recall interviews, the NHANES gathered data on participants’ food intake. A mobile examination center (MEC) served as the venue for the initial interview. Every mobile examination center had a nutritional interview room with a common set of measurement guidelines. The second dietary recall interview took place over the phone three to ten days later ( 29 , 30 ). Section title: Measurement of CDAI Educational score: 4.036668300628662 Domain: biomedical Document type: Study Language: en We used the average of two measurements to minimize bias and increase the reliability of the results. A modified version developed by previous researchers was utilized to calculate each participant’s CDAI ( 31 ). Zinc, selenium, carotenoids, and vitamins A, C, and E are the six dietary antioxidants that make up CDAI. We standardized each micronutrient by subtracting the mean of the six dietary vitamins and minerals and dividing by the total standard deviation to calculate a Z-score. Their Z-scores were then summed to obtain the composite value of CDAI. The following is the calculating formula: Section title: Evaluation of UI Educational score: 3.7076528072357178 Domain: biomedical Document type: Study Language: en In MECs (Mobile Examination Centres), only those who were ≥ 20 years old answered questions on urine incontinence. “During the past 12 months, have you leaked or lost control of even a small amount of urine with activity like coughing, lifting, or exercise?” asked participants, if they selected “yes.” grouped under “stress UI.” A positive response to the question, “During the past 12 months, have you leaked or lost control of even a small amount of urine with an urge or pressure to urinate and you could not get to the toilet fast enough?” formed the basis for the definition of “urgency UI.” If a participant answered “yes” to both the stress and urgency UI questions, they were categorized as mixed UI participants. Section title: Evaluation of covariate Educational score: 3.067700147628784 Domain: biomedical Document type: Study Language: en The following potential confounding variables were selected for this investigation based on prior research findings that may affect UI results. These variables included demographics, medical history, physical examination findings, and personal circumstances ( 6 , 8 , 32–35 ). Race, age, education, PIR, smoking status, alcohol use, diabetes, hypertension, total cholesterol levels, and exercise status were all considered categorical factors. Section title: Evaluation of covariate Educational score: 3.0164947509765625 Domain: biomedical Document type: Study Language: en We then grouped these covariates. Two groups were formed from the participants according to their age, which was fifty. The age range of the first group was under 50, whereas the age range of the second group was over or equal to 50. Three categories—below high school diploma, high school diploma, and above high school diploma—were used to classify people’s educational positions. PIR < 2 was classified as low PIR, and PIR ≥ 2 as high PIR. We divided BMI into three groups: the first group was less than 25, the second group was greater than or equal to 25 and less than 30, and the third group was less than 30. When asked if they smoked, participants were divided as smokers if they replied “yes” (SMQ020). Answer the query “In the past 12 months, on those days that you drank alcoholic beverages, on average, how many drinks did you have?” to distinguish between drinkers and non-drinkers. Those who had less than 12 drinks were categorized as non-drinkers, while those who had more than or equal to 12 drinks were considered drinkers. Section title: Evaluation of covariate Educational score: 4.040853500366211 Domain: biomedical Document type: Study Language: en Those who responded “yes” to the inquiry “Have you been told you have hypertension?,” those who were taking antihypertensive medication, and those whose average of three measurements of systolic blood pressure (BP) was ≥140 mmHg and whose average of three measurements of diastolic blood pressure (BP) was ≥90 mmHg were all considered patients with hypertension. Participants were considered to have diabetes if they answered “yes” when asked if they had diabetes or if they used glucose-lowering drugs or insulin. Meanwhile, their fasting blood glucose (≥126 mg/dL) and glycosylated haemoglobin (≥6.5%) were used to diagnose diabetes. Total cholesterol <240 mg/dL was defined as a low cholesterol level and ≥ 240 mg/dL was defined as a high cholesterol level. Section title: Statistical analysis Educational score: 3.115464210510254 Domain: biomedical Document type: Study Language: en During the processing phase, NHANES sample weights were applied to guarantee the study population’s national representation. The baseline qualities were explained after the participants were classified according to the four CDAI categories. Categorical variables, expressed as weighted percentages (%), were compared using a chi-square test. Continuous variables were compared using weighted linear regression, and the results were shown as mean (± standard deviation). Section title: Statistical analysis Educational score: 3.9828803539276123 Domain: biomedical Document type: Study Language: en To reduce confounding bias, we performed PSM processing based on UUI results. To ensure that the distribution of covariates was comparable between the UUI and non-UUI groups, we matched for the following factors: age, smoking, alcohol consumption, diabetes, hypertension, and total cholesterol ( 36–40 ). Propensity score matching (PSM) was carried out 1:1 using the R Software “MatchIt” program. The sample population was re-examined after PSM in order to validate the findings. Section title: Statistical analysis Educational score: 4.092207908630371 Domain: biomedical Document type: Study Language: en To determine whether there were any noteworthy trends or differences, a univariate analysis was performed. The study then developed three adjustment models in order to use multivariate regression analysis to examine the relationships between CDAI and UUI. Model 1 was the baseline model that did not alter any covariate variables. Race, age, education, and PIR were added to model 2. Model 3 included BMI, diabetes, hypertension, smoking, alcohol consumption, total cholesterol level, moderate activity, and vigorous activity on the basis of Model 2. A generalized additive model (GAM) was then developed to validate the dose–response relationship. A threshold effect analysis was first performed. Then, restricted cubic spline (RCS) and smooth curve fitting were used to describe the dose–response relationship between CDAI and UUI. The study used RCS to investigate whether there was a nonlinear association between CDAI and UUI, with the node set to 3. Smooth curve fitting was performed under the fully modified model. After obtaining the threshold value, the distribution of CDAI in the population was made into a violin plot for the next analysis. Section title: Statistical analysis Educational score: 3.7995264530181885 Domain: biomedical Document type: Study Language: en We then categorized the population according to the thresholds and then regressed the CDAI and its components on the UUI separately to explore whether certain elements had a greater effect. Then, using subgroup analysis, the stratified association between CDAI and UUI was examined. Additionally, interaction tests were performed to assess the way in which relationships between subgroups interacted. Section title: Statistical analysis Educational score: 2.3297336101531982 Domain: biomedical Document type: Study Language: en For all statistical studies, R (version 4.4.0) was utilized. When the two-sided p -value was less than 0.05, it was deemed statistically significant. Section title: Population characteristics Educational score: 3.837707996368408 Domain: biomedical Document type: Study Language: en Based on the screening criteria, a total of 7,735 eligible participants from NHANES 2011–2018 were selected . Among them, 1,247 had UUI and 6,488 did not. The weighted estimates for the baseline characteristics of the study population are shown in Table 1 . Study participants with a higher CDAI were found to be more heavily represented under the age of 50. In addition, those with higher levels of CDAI were more likely to be non-Hispanic white, more educated, have a higher PIR, lower smoking rates, lower alcohol consumption rates, lower body mass index, moderate exercise intensity, lower probability of having a history of diabetes and hypertension, and lower total cholesterol levels than those with lower levels of CDAI. Section title: Population characteristics Educational score: 2.860914468765259 Domain: biomedical Document type: Study Language: en We found that differences in CDAI levels differed significantly in the presence or absence of UUI and that participants with UUI had lower CDAI levels. However, no significant CDAI level differences were observed in SUI and MUI. Section title: Population characteristics Educational score: 2.9085123538970947 Domain: biomedical Document type: Study Language: en Afterwards, the CDAI distribution was analysed and visualized as a violin plot . Many people have CDAI values clustered in the lower range, even less than 0 in a large proportion, while the number of people with higher antioxidant indices is smaller (median CDAI: 0.776). Section title: Population characteristics Educational score: 4.090606212615967 Domain: biomedical Document type: Study Language: en We then balanced the effect of potential confounders associated with UUI through propensity score matching (PSM) analysis. In this investigation, a 1:1 PSM analysis was conducted. The standardized mean difference is visualized in Supplementary Figure S1 , and the distributional balance plot of PSM is shown in Supplementary Figure S2 . Following PSM, 1702 participants were enrolled in the study; 851 of them had UUI, while the remaining individuals did not . The weighted essential attributes of the research subjects were displayed in Supplementary Table S1 following PSM. Differences in variables between the two groups were managed to some degree. After PSM, age, race, education, PIR, hypertension, total cholesterol, vigorous activity, and UUI were found to be significantly associated with CDAI. Section title: Univariate analysis of UUI Educational score: 3.8776533603668213 Domain: biomedical Document type: Study Language: en Preliminary exploration of pre- and post-PSM data with a univariate analysis of UUI. We found the following: prior to PSM, UUI was positively associated with age ≥ 50 years, body mass index ≥25, non-Hispanic black, hypertension, diabetes mellitus, and high cholesterol. In addition, UUI was negatively associated with other races, high school and higher education, high PIR, nonsmokers, vigorous activity, and Q2 and Q4 in the CDAI quartiles. After PSM, UUI was not associated with covariates such as age, high school education, 25 ≤ BMI < 30, smoking, hypertension, diabetes mellitus, level of exercise, and cholesterol level. Section title: Univariate analysis of UUI Educational score: 3.7625391483306885 Domain: biomedical Document type: Study Language: en After PSM, only BMI ≥ 30 was positively associated with UUI. In contrast, other races, higher than high school education level, high PIR, and Q2 in CDAI were negatively associated with UUI ( Table 2 ). We found that Q2 in CDAI was negatively correlated with UUI both before and after PSM ( p = 0.007 and p = 0.004). Section title: The relationships between CDAI and UUI Educational score: 3.843442440032959 Domain: biomedical Document type: Study Language: en To further explore the relationship between CDAI and UUI, a weighted logistic regression analysis of the three models was conducted . The CDAI was converted (Q1-4; Q1 was used as a reference point) and examined by quaternity. In Model 1, no variables have been added. In Model 2, race, Age, education, and PIR adjustments were made. Model 3 was built using Model 2, modified to account for BMI, smoking, alcohol consumption, hypertensive disease, diabetes, total cholesterol, moderate activity, and vigorous activity. Section title: The relationships between CDAI and UUI Educational score: 4.08924674987793 Domain: biomedical Document type: Study Language: en In the pre-PSM analysis, only Q2 ( p = 0.007) and Q4 ( p < 0.001) were negatively correlated with UUI in the crude model, and this relationship was lost in the other models. In the analysis following the PSM, a negative correlation between Q2 and UUI was found in all three models ( p = 0.004 in the crude model, p = 0.034 in model 2, and p = 0.028 in model 3). Section title: Connectivity between CDAI and UUI in terms of dose–response Educational score: 4.121166229248047 Domain: biomedical Document type: Study Language: en In the threshold effect analysis, we found no linear relationship between CDAI and UUI either before or after PSM ( p = 0.383 and p = 0.483). Moreover, the negative correlation between CDAI greater than or less than the K value and UUI was not significant before PSM ( p = 0.086 and p = 0.341). After PSM, CDAI was negatively correlated with UUI when CDAI was <K value ( p = 0.027), and the relationship was not significant when it was > K value ( Table 3 ). Section title: Connectivity between CDAI and UUI in terms of dose–response Educational score: 4.136436939239502 Domain: biomedical Document type: Study Language: en Figure 4 displays the results of the dose–response relationship for the Restricted Cubic Spline (RCS) (node setting of 4 before and after PSM). Under the fully adjusted model, CDAI and UUI prevalence showed a nonlinear relationship before and after PSM (nonlinear p of 0.010 before PSM, nonlinear p of 0.007 after PSM). At OR = 1, the reference values for CDAI before and after PSM were 0.776 and 0.523, respectively. The overall relationship between CDAI and UUI was significant ( p for overall = 0.024 before PSM and p for overall = 0.018 after PSM). Section title: Segmented regression analysis and sensitivity analysis Educational score: 3.2969186305999756 Domain: biomedical Document type: Study Language: en The K value after PSM (K = 0.523) was used to distinguish the two groups of people and the multiple regression analysis of CDAI and UUI was performed before and after PSM, respectively. The nutrient elements of each component in the CDAI<0.523 group were subjected to sensitivity analysis ( Supplementary Table S2 ) and visualized . Section title: Segmented regression analysis and sensitivity analysis Educational score: 4.094314098358154 Domain: biomedical Document type: Study Language: en The negative correlation with UUI was not significant for CDAI ≥0.523 either before or after PSM ( p = 0.323 before PSM, p = 0.278 after PSM). However, at a CDAI less than 0.523, the CDAI was negatively correlated with UUI incidence both before and after PSM (OR = 0.92, p = 0.03 before PSM and OR = 0.91, p = 0.033 after PSM). Moreover, among the components, only the z-score of zinc showed a relatively significant negative correlation with UUI (OR = 0.72, p = 0.002 before PSM and OR = 0.74, p = 0.02 after PSM), and no significant correlation was found between the rest of the components and the incidence of UUI. Section title: Segmented regression analysis and sensitivity analysis Educational score: 4.110079288482666 Domain: biomedical Document type: Study Language: en To further explore the relationship between CDAI and zinc with UUI, we performed RCS plots for CDAI and zinc, respectively, under the fully adjusted model with the node setting of 3 before PSM . Neither the CDAI nor the z score of Zinc showed a nonlinear relationship with UUI (CDAI, nonlinear p of 0.754, Zinc, nonlinear p of 0.262). At OR = 1, the reference values were − 1.672 and − 0.287, respectively. The overall p -value for the RCS plotted for the relationship between CDAI and UUI was 0.042. Section title: Subgroup analysis between CDAI and incidence of UUI Educational score: 4.089888572692871 Domain: biomedical Document type: Study Language: en The study stratified the first subsection by race, age, education, PIR, body mass index, diabetes mellitus, hypertension, whether or not they smoked, whether or not they drank alcohol, total cholesterol, and moderate and vigorous activity ( Table 4 ). Analyses were statistically significant regardless of pre- and post-PSM ( p = 0.03 before PSM and p = 0.033 after PSM). Before and after PSM, CDAI was significantly negatively associated with UUI among those whose race was other Hispanic, higher than high school education, high PIR, non-drinkers, diabetics, and high cholesterol levels. In addition, after PSM, CDAI was negatively associated with UUI among those aged ≥50 years. Moreover, race played a significant moderating role in the effect after PSM, p for interaction = 0.043. It suggests a significant difference between racial subgroups. Apart from this, no significant interaction effects were found in other subgroups regardless of before or after PSM, suggesting that our results apply to almost all types of male populations. Section title: Investigating possible mechanistic connections Educational score: 3.976135015487671 Domain: biomedical Document type: Study Language: en Based on Figdraw, an intuitive diagram is presented in Figure 7 . We discuss the mechanisms by which CDAI affects the prevalence of UUI in Part 4. In the figure, the green arrows represent typical foods rich in the Composite Dietary Antioxidant Index (CDAI), which are beneficial for reducing the risk of UUI. The red arrows indicate biological mechanisms discussed in the article that contribute to the occurrence of UUI. These include markers of inflammation and oxidative stress: CRP (C-reactive protein), IL-1β, IL-6, IL-8 (cytokines), ROS/RNS (reactive oxygen and nitrogen species), and MCP-1 (monocyte chemoattractant protein-1). Other involved factors include Rho (Ras homologous), parasympathetic nerves, detrusor overactivity (DO), and peptidergic/sensory nerves (For more details, please refer to the discussion section). Section title: Discussion Educational score: 3.9496612548828125 Domain: biomedical Document type: Study Language: en In this cross-sectional study of a nationally representative sample of U.S. men, greater consumption of antioxidant micronutrients was connected to a reduced likelihood of UUI in men with subthreshold CDAI intake (K = 0.523). However, the CDAI was not significantly associated with SUI or MUI. Section title: Discussion Educational score: 3.9032485485076904 Domain: biomedical Document type: Review Language: en Overactive bladder (OAB) syndrome is frequently accompanied by UUI. Prior research has indicated that 82.9% of patients with OAB have UUI ( 41 ). The connection between inflammation and overactive bladder (OAB) has been the subject of several investigations. Increased CRP levels were consistently linked to OAB in men, according to a study that investigated people in Boston between the ages of 30 and 79 ( 42 ). The idea that inflammatory infections may be an undervalued factor in the etiology of certain OAB patients is supported by a review article that discusses important findings from recent clinical and laboratory studies, including the relationship between bladder inflammation, urinary tract infections, and OAB pathogenesis ( 43 ). Section title: Discussion Educational score: 4.376761436462402 Domain: biomedical Document type: Study Language: en Studies of the pathophysiology of urolithiasis have found that UUI involves molecular mechanisms: signalling and inflammation ( 44 ) . Increased serum C-reactive protein (CRP) levels in patients with UUI compared to non-patients have been demonstrated in several studies ( 45 ). Studies examining ischemia-modified albumin (IMA) have found that IMA levels are significantly elevated in patients with UUI compared to non-UUI patients ( 46 ). IMA levels are elevated in Systemic Inflammatory Response Syndrome (SIRS) and some inflammatory diseases. This may be related to the large number of free radicals released during the inflammatory process. Serum levels of interleukin-1β, −6 and − 8 have also been found to be elevated in UUI patients ( 47 ). IL-6 and IL-1β: elevated levels in a variety of chronic inflammatory diseases drive long-term inflammatory responses. IL-8 attracts primarily neutrophils to sites of inflammation and promotes infiltration of inflammatory cells. Urine levels of monocyte chemotactic protein 1 (MCP-1) in patients with UUI showed a trend toward higher levels compared to non-UUI patients ( 46 ). The pathophysiology of bladder dysfunction and the control of connexin expression are significantly influenced by inflammatory cytokines ( 48 ). Additionally, the relationship between local immune cells and overactive bladder parasympathetic and peptidergic/sensory innervation is mediated by inflammatory cytokines ( 49 ). Section title: Discussion Educational score: 4.191517353057861 Domain: biomedical Document type: Study Language: en A “vicious cycle” can be created when oxidative stress triggers an inflammatory response by triggering inflammatory signalling pathways, which can further intensify oxidative stress. In an acute inflammatory response, neutrophils and macrophages kill pathogens by releasing large amounts of ROS through an oxidative burst (respiratory burst). The accumulation of reactive oxygen species (ROS) leads to the oxidation of DNA, proteins, carbohydrates lipids, and apoptosis ( 50 ). Diet controls the plasma redox state as an external factor and shields the body from reactive oxygen and reactive nitrogen species. Scavenging oxidants and antioxidants prevent oxidative damage by preserving a stable cellular redox state ( 51 ). Exogenous intake of antioxidants may prevent urinary incontinence and bladder ischemia ( 52 , 53 ). Section title: Discussion Educational score: 4.096138000488281 Domain: biomedical Document type: Study Language: en Our research revealed that CDAI was negatively and linearly correlated with UUI in men when CDAI was below 0.523 (median CDAI: 0.776). However, this relationship lost its significance when the CDAI value was greater than 0.523. This may be related to the saturating effect of dietary antioxidants. According to a study, dietary antioxidants and depression in persons who are overweight or obese are negatively correlated. However, in the group that was overweight, saturation effects were noted ( 54 ). In the study of dietary antioxidants and coronary heart disease, a threshold effect of complex dietary antioxidants was also found ( 55 ). A study examining dietary anti-inflammation and cognitive dysfunction in older adults also found a saturating effect of complex dietary antioxidants ( 56 ). In addition, excessive vitamin C intake is positively associated with the development of urinary incontinence ( 52 ). Because of the threshold effect, antioxidants have been shown to exhibit opposite effects under certain conditions ( 57 ). Section title: Discussion Educational score: 4.063958644866943 Domain: biomedical Document type: Study Language: en More crucially, in the study of oxidative balance fractions and urinary incontinence, behavioural oxidative balance fractions were found to have a much greater effect on urinary incontinence than dietary oxidative balance fractions. And the behavioural oxidative balance score included physical activity, body mass index, alcohol consumption, and cotinine ( 58 ). All these four variables were included in our covariates, which had a significant impact on the prevalence of CDAI and UUI. As a result, at a CDAI greater than 0.523, the negative connection with UUI prevalence is no longer significant. In our stratified analyses, we found that the relationship between the prevalence of CDAI and UUI did not differ significantly in populations differing in BMI, smoking, and vigorous or moderate activity, regardless of before or after PSM. The relationship between the prevalence of UUI and CDAI was more significant only in those who did not drink alcohol. Section title: Discussion Educational score: 4.084483623504639 Domain: biomedical Document type: Study Language: en We further stratified by each covariate and found no significant interaction effects before or after PSM except for the variable race after PSM, suggesting that our findings are applicable to almost all types of male populations. In subgroup analyses, there was greater sensitivity to the protective effects of CDAI among those whose race was other Hispanic, higher than high school education level, PIR ≥2, did not consume alcohol, had diabetes, and had high cholesterol levels. An article examining racial differences in overactive bladder found that urge incontinence has a high prevalence among Hispanics ( 59 ). Alcohol intake is positively associated with lower urinary tract disorders such as OAB ( 60 ). Several studies have confirmed that the prevalence of UI is increased in poor populations and exacerbated by complex social, cultural and psychological influences ( 61 ). It has been shown that the correlation between diabetes and OAB is more significant through systemic inflammation as a mediator ( 62 ). Research on the relationship between hypercholesterolemia and UUI is still lacking, but a study in rats found that hypercholesterolemia was positively associated with detrusor overactivity (DO) ( 63 ). In summary, we have identified specific beneficiary populations most likely to benefit from increased antioxidant dietary micronutrients to reduce the prevalence of UUI. In adult males with CDAI levels below 0.523, increasing dietary antioxidant intake is associated with a decrease in the incidence of UUI. This may provide some theoretical basis for the prevention or dietary treatment of UUI in clinical as well as public health settings. Section title: Discussion Educational score: 4.183176040649414 Domain: biomedical Document type: Study Language: en In our compositional analysis, we found that the negative correlation between zinc and the incidence of UUI was more pronounced. The study found that zinc plays a significant role in antioxidant defense and inflammation regulation ( 64 ). Zinc acts as a cofactor for the antioxidant enzyme superoxide dismutase (SOD1), facilitating the conversion of superoxide radicals into less harmful molecules, thereby reducing oxidative stress and preventing cellular damage. In addition, zinc plays an important role in neural function by regulating neurotransmitter release, supporting synaptic plasticity, and protecting neurons from oxidative damage ( 65 ). Zinc regulates inflammation by modulating the production of pro-inflammatory cytokines (such as TNF- α and IL-6) and promoting an anti-inflammatory immune response, primarily through the inhibition of NF-κB activation ( 66 ). Therefore, zinc, as an important component of CDAI, plays a significant protective role in the risk of UUI. This study utilized a large, representative sample of adult males from NHANES and followed a carefully designed research protocol. To the best of our knowledge, this is the first study to explore the association between CDAI and UUI in men using a large sample size. We used univariate regression, multivariate regression, threshold effects analysis, sensitivity analysis, and subgroup analysis to enhance our understanding of their relationship. We investigated the relationship between composite dietary trace elements, which better reflect real-life scenarios, and the incidence of UUI in men. Additionally, we identified the threshold of CDAI that influences UUI in men. This threshold could be beneficial in clinical practice for preventing UUI and predicting the risk of UUI in adult men. Nevertheless, there are certain unavoidable limits to our study. First, since the study was cross-sectional, it was impossible for us to establish causality. Therefore, future longitudinal studies or randomized controlled trials are needed to better understand the causal relationship. Second, the potential bias easily introduced by dietary data from interviews leads to potentially biased results. Finally, we are unable to determine the residual confounding effects that may arise from unmeasured factors. Therefore, more research is needed in the future to address these limitations and provide further insights. Section title: Conclusion Educational score: 4.049173355102539 Domain: biomedical Document type: Study Language: en This study sought to increase understanding of the role antioxidant diets play in UUI prevalence among men. The results showed that when CDAI was below the threshold, the incidence of UUI was negatively correlated with CDAI. Therefore, CDAI can be used to predict the risk of UUI in men and guide the prevention of UUI in men. Men with lower dietary antioxidant micronutrient intake (approximately half of the adult male population in the United States) experience a reduced risk of UUI as their dietary antioxidant intake increases. | Review | biomedical | en | 0.999997 |
PMC11695337 | Section title: 1 Introduction Educational score: 4.280424118041992 Domain: biomedical Document type: Study Language: en Working memory (WM) is crucial for preparing and organizing goal-directed behaviors, with its functions of storing and manipulating incoming information. This process is capacity-limited, demanding a balance between stability (preserving the WM content from irrelevant information) and flexibility (updating WM with relevant information) . It involves three temporal subprocesses: encoding, maintenance, and retrieval. Over the years, various models of WM functioning have been created, but the new ones are based more on the dynamics of the ongoing processes. The dynamic-processing model of working memory assumes that relevant information is retained by creating representations through recurrent activity and/or strengthening the synaptic weights between neurons. These processes are very dynamic and context-dependent. One of the leading hypotheses in functional neuroimaging studies on false memory retrieval is the sensory reactivation hypothesis. It suggests that memory retrieval reactivates the processes from the encoding stage, with true memories involving activation of sensory areas . However, recent findings indicate that content representations during retrieval differ from those during encoding. Memory retrieval appears to be more of a constructive and dynamic process involving the frontoparietal cortex, rather than just the reactivation of the sensory cortex as suggested by the sensory reactivation hypothesis . To investigate the dynamic of working memory processes, we designed an fMRI experiment with four visual working memory tasks: two with visuospatial and two with verbal stimuli based on the Deese–Roediger–McDermott (DRM) paradigm , and additionally resting-state procedure. The DRM paradigm is widely used in memory research, as it separates the WM subprocesses like encoding and retrieval. Section title: 1 Introduction Educational score: 4.215743064880371 Domain: biomedical Document type: Review Language: en In recent years, a variety of innovative methods have been applied to the analysis of fMRI data. These include machine learning algorithms, nonlinear time series analysis, and complex network methodologies . Significant research has focused on utilizing machine learning techniques and neural networks for fMRI data analysis. The main idea behind this approach is to analyze the neuroimaging data not from the point of view of the single voxel, but by identifying the patterns of neural activity over many brain areas. Thus, classification based on advanced computational algorithms is one of the most efficient methods for identifying neural activity and extracting the complex relationship between the experimental conditions and spatial-temporal patterns of brain responses measured by the fMRI technique . However, the machine learning algorithms and neural network types must be matched to the problem investigated to obtain statistically reasonable results, which is not a trivial problem due to the variety of computational methods, e.g., linear discriminant analysis (LDA), support vector machines (SVM), random forests (RF), neural networks classifiers and many others. Therefore, the performance of machine learning algorithms has been examined in many neuroscience studies, including research related to the classification and diagnosis of Alzheimer's, Huntington and schizophrenia disease , cognitive functions , sleep studies and conscious visual perceptions . Section title: 1 Introduction Educational score: 4.067363262176514 Domain: biomedical Document type: Study Language: en The goal of this paper is twofold. First, we would like to apply some of the most commonly used machine learning algorithms and neural networks in the study with working memory and verify their effectiveness in classifying the tasks (recognition between the visuospatial and verbal stimuli) and phases of the experiments (encoding and retrieval). In this study, we considered a set of linear and nonlinear classifiers and a residual neural network. We also regarded two kinds of data organizations, i.e. in the temporal-spatial form and single-time points from each observation. Secondly, based on the results from classification experiments, we would like to determine the brain regions that are the most important for classifiers; thus, important from the point of view of information processing in the brain. To this end, we proposed a novel algorithm and compared the results with the outcomes from the literature. Section title: 2.1 Data description Educational score: 4.053394317626953 Domain: biomedical Document type: Study Language: en Functional magnetic resonance imaging (fMRI) data from four short-term memory tasks and a resting-state procedure were analyzed. Based on questionnaires and genotyping of the PER3 gene, 66 participants out of 5,354 volunteers were selected to perform the tasks in the MR scanner during two sessions: morning and evening. After further data quality control, 58 participants were included in the analysis. The order of the sessions as well as the versions of the tasks (there were two equivalent versions of each task) were counterbalanced between participants. The experiment was conducted on one day (if the morning session was the first) or two days (if the evening session was the first). Participants spent the night before or between sessions in the room located in the laboratory, and their quality of sleep during that night and the week preceding the experiment was controlled using actigraphs. Section title: 2.1 Data description Educational score: 4.005084991455078 Domain: biomedical Document type: Study Language: en The short-term memory tasks were based on Deese-Roediger-McDermott (DRM) paradigm , which allows separating the encoding and retrieval processes, and is dedicated to studying short-term memory distortions. Two tasks evaluated the perceptual similarity (focusing on global, GLO, and local, LOC, information processing of abstract objects), and the remaining two the verbal similarity (semantic, SEM, and phonological, PHO, task). More specifically, in GLO task, the stimuli were abstract figures requiring holistic processing; in LOC task, the objects required local processing and differed in one specific detail. On the other hand, in SEM task, participants had to remember four Polish words matched by semantic similarity, and in PHO task, matched by phonological similarity. Section title: 2.1 Data description Educational score: 4.035684585571289 Domain: biomedical Document type: Study Language: en In each task, the goal of a participant was to memorize the memory set (encoding phase, 1.2–1.8 s), and then (after a mask or distractor) recognize if the currently displayed stimulus, called probe, was present in the preceding set (retrieval phase, 2 s). There were three possible conditions of the probe: positive (the stimulus was the same as presented in the encoding phase), negative (the stimulus was completely different) or a lure (the stimulus resembled those presented in the memory set). Participants were asked to answer with the right-hand key for “yes” and the left-hand key for “no” responses. Then, after intertrial interval (on average 8.4 s), a fixation point (450 ms) and a blank screen (100 ms), the new memory set was presented on the screen. Each task had 60 sets of stimuli followed by 25 positive probes, 25 lures, and 10 negative probes. The examples of experimental tasks are depicted in Supplementary Figure 1 . Section title: 2.1 Data description Educational score: 2.5957162380218506 Domain: biomedical Document type: Study Language: en In the resting-state procedure (REST), participants were instructed to lie in the scanner with their eyes open and not to think about anything in particular. They were not involved in any cognitive process. Participants' awakeness was monitored using an eye-tracking system . Section title: 2.1 Data description Educational score: 4.109776020050049 Domain: biomedical Document type: Study Language: en Structural and functional data were collected on a 3T scanner Skyra (Siemens Magnetom, Erlangen, Germany) in Małopolska Center of Biotechnology in Kraków, Poland, with a 64-channel head coil. For tasks, 709 volumes (for GLO, LOC, and SEM), and 736 volumes (for PHO task) with a T2*-weighted echo-planar sequence were acquired. For the resting-state procedure, 335 volumes with a gradient-echo single-short echo planar imaging sequence were acquired. The following scanning parameters were used: TR = 1,800 ms, TE = 27 ms, flip angle = 75°, FOV = 256 mm, voxel size: 4 × 4 × 4 mm). Structural data were acquired for each participant using a T1-weighted MPRAGE sequence with isotropic voxels (1 × 1 × 1.1 mm) using the following parameters: 256 × 256 mm matrix, 192 slices, TR = 2,300 ms, TE = 2.98 ms. Stimuli were projected on a screen behind a participant's head. The participants viewed the screen in a 45° mirror fixated on the top of the head coil. Section title: 2.2 Data preprocessing Educational score: 4.099294662475586 Domain: biomedical Document type: Study Language: en The flow chart in Figure 1 summarizes data preprocessing steps. Following the paper that introduced the dataset , we performed the preprocessing using the Statistical Parametric Mapping software package (SPM12, Welcome Department of Imaging Neuroscience, UCL, London, United Kingdom) implemented in MATLAB (Mathworks, Inc., MA, United States). Scans were slice-time corrected, realigned by inclusion of field maps, co-registered, and normalized to the EPI template in Montreal Neurological Institute (MNI) stereotactic space with a voxel resolution 3 × 3 × 3 mm. Then, data were spatially smoothed using a Gaussian kernel of FWHM 6 mm, covariates like motion parameters, mean signal, white matter, and CSF were removed by linear regression. The signal was then filtered with a 0.01–0.1 Hz filter, detrended, and despiked. Section title: 2.2 Data preprocessing Educational score: 4.144877910614014 Domain: biomedical Document type: Study Language: en The signals were then averaged within 116 brain Regions of Interest (ROIs) using the Automated Anatomical Labeling (AAL1) brain atlas . For a given experimental session, they formed a data matrix with 116 rows corresponding to the ROIs and the number of columns corresponding to the length of the time series. Further processing steps follow : for each session, we extracted all data segments (i.e., columns) related to the encoding or retrieval phase; for each phase, the segments were then concatenated in order of appearance. The segments started at the stimulus onset and were 10s long (6–7 TRs) each, which means that the encoding segments encompassed the presentation of the distractor and the retrieval segments encompassed the inter-trial interval. For a given session, the concatenation of segments from all 60 stimuli resulted in a 400-TR-long time series. Data for the resting state with eyes open were preprocessed similarly to the task data. First, in the absence of stimuli that would set the position of the segments, 400-TR long series were randomly chosen. Then, they were divided into consecutive, non-overlapping segments of 10s. Then, these segments were randomly shuffled and concatenated. Section title: 2.3 Classification tasks Educational score: 2.37795352935791 Domain: biomedical Document type: Study Language: en We create eight classification problems using the data described in Section 2.1, four for each of the two phases of encoding (ENC) and retrieval (RET). Their variants are binary or 4-class problems depending on whether we group similar stimuli or not, e.g. global and local stimuli are both graphical and can be grouped into a single class. They can be further complicated by adding the resting state as an additional class, resulting in a 3- or 5-class classification. Table 1 lists the resulting experimental setups. Section title: 2.3 Classification tasks Educational score: 1.9852226972579956 Domain: biomedical Document type: Study Language: en For each of the listed setups, we randomly split the available data into training and testing subsets using a ratio of 90:10. Section title: 2.3 Classification tasks Educational score: 2.767738103866577 Domain: biomedical Document type: Study Language: en In all non-neural classifiers, a sample of input data always has the dimensions of 116 × 1 (number of brain regions times one time point). We also conduct additional experiments taking into account the time dimension of the data, where we train neural networks on samples of dimensions 116 × 6 (sequences of six time points). We elaborate on this experiment in Section 5.2. Section title: 3 Models Educational score: 2.5069491863250732 Domain: biomedical Document type: Study Language: en We have conducted the fMRI classification by dividing machine learning methods into classical linear and non-linear discriminators, and neural networks. In total, ten classifiers were compared in the study. Section title: 3.1.1 Ridge classifier Educational score: 2.2894797325134277 Domain: other Document type: Other Language: en Ridge classifier was proposed in Hoerl and Kennard . The method addresses the problem of parameter estimation for multi-collinear independent variables. Ridge achieves that by adding a penalty term L2, which is equal to the square of the coefficients. However, while the L2 regularization minimizes coefficients, it never reduces them to zero . Section title: 3.1.2 Logistic regression Educational score: 2.350476026535034 Domain: biomedical Document type: Other Language: en Logistic regression is another linear estimation algorithm that was tested in the study. Unlike the Ridge Classifier, the logistic regression uses a cross-entropy loss function to output the probability for the classification . Section title: 3.1.3 Support vector machines using stochastic gradient descent (SGD) Educational score: 1.9430363178253174 Domain: other Document type: Other Language: en Loss optimization in linear models can also be performed using stochastic gradient descent SGD. It attempts to discover the gradients of the cost function for a random selection of data points. By conducting this operation, stochastic gradient descent can lead to much faster convergence of the algorithm. This timing advantage is crucial in the case of Support Vector Machines, which tend to build complex hyperplanes . Section title: 3.1.4 Gaussian naive Bayes Educational score: 2.357313394546509 Domain: other Document type: Other Language: en Gaussian naive Bayes is a subset of the Naive Bayes models. The model assumes a Gaussian distribution of the data and a lack of feature dependencies within it. Due to its simplicity, the classifier often performs well on highly dimensional data. It can converge faster than discriminative algorithms such as Random Forest or Logistic Regression. The Gaussian Naive Bayes can also serve as a baseline due to its probabilistic nature . Section title: 3.1.5 Linear discriminant analysis (LDA) Educational score: 2.4517223834991455 Domain: biomedical Document type: Other Language: en LDA is another linear method that was evaluated in the study. LDA operates in two stages. First, it extracts all the feature values linearly. Then it uses those mappings and attempts to linearly separate classes by presenting points of opposite classes as far as possible from each other . Section title: 3.2 Non-linear classifiers Educational score: 2.943187713623047 Domain: biomedical Document type: Other Language: en In contrast to the discriminators utilizing linear functions, non-linear classifiers attempt to match the data by minimizing functions that do not share regular slopes. This approach allows for creating more strict boundaries between classified data points, hence increasing the goodness of a fit. However, at the same time, this can lead to non-linear classifiers overfitting. Section title: 3.2.1 Quadratic discriminant analysis (QDA) Educational score: 2.6051104068756104 Domain: biomedical Document type: Other Language: en Unlike LDA, which relies on the assumption of linear divisibility of the feature values, Quadratic Discriminant Analysis, uses non-linear attributes to separate data points. This proves to be generally more accurate, especially in the problems with high dimensionality . Section title: 3.2.2 Random forest classifier Educational score: 2.4549617767333984 Domain: other Document type: Other Language: en Random forest classifier is a model composed of a collection of decision trees. In simple terms, decision trees can accept both numeric and categorical inputs to build sets of rules. Those rules are distributed throughout the trees. The data input can then flow in a top-to-bottom way and be filtered to produce respective outputs and classification results. Random forests use sets of such rules to provide even more accurate classification boundaries . Section title: 3.2.3 Light gradient boosting machine (LGBM) Educational score: 1.6493995189666748 Domain: other Document type: Other Language: en Over the last few years, boosting methods have been receiving more attention as their performance shows improvement over simple tree-based approaches. The reason for this phenomenon originates in the architecture of the boosted models. Boosting combines multiple models that are built iteratively, rather than in parallel. This ensures that each consecutively built algorithm attempts to minimize the errors made by previous models . Light gradient boosting is a special case of such an application, as it proves to outperform most of the other machine learning algorithms in a variety of benchmarks . Section title: 3.3.1 Dummy classifier Educational score: 2.2589194774627686 Domain: biomedical Document type: Study Language: en To assess the reliability of the performed experiments, We created a dummy classifier whose sole purpose was to create a baseline for the other methods. The dummy classifier was fit in such a way that it ignored the input features and relied only on the distribution of the classified classes in making predictions. Section title: 3.4 Neural networks Educational score: 4.0320587158203125 Domain: biomedical Document type: Study Language: en One of the motivations for using deep neural networks, specifically the ones employing convolutional layers (like convolutional and residual networks), is the ability to process spatial data. In our case, the original BOLD data can be considered as having two dimensions—the first corresponds to the spatial distribution of the ROI, and the second corresponds to the temporal character of the time series. Each data sample consists of up to six discrete measurements, one TR apart, corresponding to the duration of the encoding/retrieval phases. We hypothesize that having the data in the temporal-spatial form and a model processing this data structure can yield better results than using single-time observations. Section title: 3.4.1 Layered convolutional network (CNN) Educational score: 3.991347312927246 Domain: biomedical Document type: Study Language: en The 1D convolution is often used for processing temporal data due to its ability to effectively handle sequential information in a channel-wise manner . The CNN architecture we use comprises two 1D convolutional layers (all the convolutional layers use filters of size 3), 1D maximal pooling, another 1D convolution, a linear layer with dropout, and the final softmax layer to calculate class probabilities. Crucially, all the convolutions move along the temporal dimension. This setup allows interactions between any subset of brain regions. Section title: 3.4.2 Residual network (ResNet) Educational score: 4.139256477355957 Domain: biomedical Document type: Study Language: en Residual networks (ResNets) are a more powerful neural network type, in our case also utilizing convolutions. They consist of an input convolution, followed by several residual blocks of layers, depending on the network depth. Each of the blocks comprises two convolutional operations (3 × 3 filters), batch normalization , and a shortcut connection (1 × 1 filters). In the final layers, average pooling is performed, and a linear operation computes logits for each label. We utilize 2D convolutions to capture both spatial and temporal interactions between different ROIs. Taking inspiration from Li et al. and Suk et al. , we organize the ROIs in a structured format within a 2D matrix. This enables our network to effectively learn interactions between brain regions neighboring in the AAL atlas (typically, spatially adjacent or contralateral), allowing for the extraction of both local spatial relationships and temporal patterns simultaneously. Figure 2 shows a schematic overview of a typical architecture of a residual network. Section title: 4 Methods Educational score: 1.615457534790039 Domain: other Document type: Study Language: en This section expands on two out of the several models described in Section 3: LGBM and ResNet. This selection was made after the initial results, where LGBM and ResNet models proved to be the most promising ones (LGBM scores were comparable or higher than all other models, but for ResNet in two encoding experiments). Consequently, we included here the hyperparameter tuning and feature explanation procedures of the two models only. Section title: 4.1.1 LGBM hyperparameter tuning Educational score: 2.269442319869995 Domain: biomedical Document type: Study Language: en In our preliminary calculations, the tested machine learning algorithms were capable of resolving the complexity of correlations and differences within our sample of the fMRI data with varying accuracy. Therefore, we decided to pick the most promising model—LGBM—and improve it with hyperparameter tuning . Section title: 4.1.1 LGBM hyperparameter tuning Educational score: 2.676297903060913 Domain: biomedical Document type: Study Language: en To perform the parameter searches, we used Optuna, a state-of-the-art hyperparameter optimization framework . We conducted 100 repetitions of each experiment, such as ENC2 or RET4. Within each repetition, there were 100 hyperparameter searches, and optional pruning of trials with unpromising initial results. This procedure reduces the bias of human-produced parameters and can be utilized again, even if the data distributions have changed . The parameters used for the experiments can be seen in Table 2 . Henceforth, we refer to the hyperparameter-tuned Light Gradient Boosting model as “tuned LGBM.” Section title: 4.1.2 Neural network training and hyperparameter tuning Educational score: 2.9348995685577393 Domain: biomedical Document type: Other Language: en In neural network experiments, each model is trained from scratch for up to 100 epochs. Early stopping was employed to finish the training process before epoch 100 if no improvement in validation loss was recorded for five epochs in a row. All networks were trained using the Adam optimizer together with a scheduler to reduce learning rate on loss function plateaus. Section title: 4.1.2 Neural network training and hyperparameter tuning Educational score: 2.9965527057647705 Domain: biomedical Document type: Study Language: en We perform a hyperparameter search for each task, where each task deals with a different data collection phase (either encoding or retrieval ) and a varying number of classes—depending on whether the labels are simplified, by joining similar stimuli, and the addition of resting state data. Table 3 lists hyperparameters and their value ranges searched during training for ResNets and, similarly, Table 4 lists the hyperparameters for the 1D convolutional model. Section title: 4.2.1 ROI importance estimation for LGBM Educational score: 3.668098211288452 Domain: biomedical Document type: Study Language: en In addition to obtaining classification metrics for the experiments carried out, we were also interested in determining which particular ROIs were the most vital features for the classifier. To this end, we extracted ROI importance scores from the LGBM models using the default LGBM feature importance estimation method , i.e. the number of splits. The method, applicable to any tree-based model, counts the number of times a given feature (ROI activations) was used to split the data to grow the decision tree. We report the results of the tuned LGBM in the Supplementary material . Section title: 4.2.1 ROI importance estimation for LGBM Educational score: 4.003711700439453 Domain: biomedical Document type: Study Language: en Unfortunately, such scores—as many others —cannot account for possible correlations between the features. This lack could result, for example, in a pair of equally informative and highly correlated features being recognized as an important and an unimportant one; if, however, the former was removed from the data, the latter would take over its importance. To alleviate this problem, we performed an additional feature pruning procedure as described in the Algorithm 1 : at each step, (i) the most important feature was removed from the training set, (ii) importance scores of the remaining features were recomputed, (iii) to correct for the decreasing number of features, the mean of importance across the remaining features was subtracted from the importance of each individual feature. The process finished when all features have been removed. To obtain the final corrected score, such demeaned scores were averaged over all the steps in which ROI was present in the model. The process is illustrated in Figure 3 . Given the significant computational time and resources required for feature pruning, we limited the validation to untuned LGBM models. Section title: 4.2.2 ROI importance estimation for ResNets Educational score: 1.6789854764938354 Domain: other Document type: Other Language: en By design, deep neural networks are black-box models, i.e., there is no simple way to obtain insight about why they generated a certain prediction. As opposed to a linear model, in deep neural networks the input representation is non-linearly transformed multiple times. Because of that, to extract interpretable knowledge about a network's decision process, we have to resort to various heuristics. Section title: 4.2.2 ROI importance estimation for ResNets Educational score: 4.015255928039551 Domain: biomedical Document type: Study Language: en Our approach, outlined in Algorithm 2 , is inspired by methods that perturb the input data during evaluation and measure the decrease in performance . More formally, given a trained fixed ResNet model f and a validation set X = ( x 1 , x 2 , …, x n ), we sample batches from the validation set, X B = ( x B 1 , …, x Bk ) and perturb in them a subset R of ROIs X ~ B = P ( X B , R ) . Both the perturbed batch X ~ B and the original X B are then passed to the model and have their predictions compared. Finally, differing predictions contribute to the scores collected for each perturbed ROI in R . Section title: 4.2.2 ROI importance estimation for ResNets Educational score: 3.9685070514678955 Domain: biomedical Document type: Study Language: en In more detail, the perturbation function P (·, R ) is assumed to change only a subset of the input space so that this modified subset of features would now be unusable by the model f . We choose a zeroing perturbation function P ∅ ( x, R ), which sets to 0 a subset of feature representation of x — in our case, a subset of k ROIs chosen uniformly at random for each batch X B . For each original data point x ∈ X B and its perturbed counterpart, x ~ ∈ X ~ B we compare their prediction p = f ( x ) and p ~ = f ( x ~ ) and note if they differ. If they do, we add one to the batch score Section title: 4.2.2 ROI importance estimation for ResNets Educational score: 2.4251596927642822 Domain: biomedical Document type: Other Language: en In other words, the score s is a percentage of predictions in the perturbed batch differing from the predictions in the original batch. We store it in a buffer, for each zeroed ROI r ∈ R in this batch. The per-batch scores s are collected over multiple iterations and batches for each r ∈ ROIs, and their average is the final importance score S [ r ]. Section title: 4.2.2 ROI importance estimation for ResNets Educational score: 3.8761160373687744 Domain: biomedical Document type: Study Language: en The procedure was run for N = 10,000 iterations, with k = 12 out of 116 ROIs zeroed in each batch – the choice was arbitrary, but it involves a reasonable trade-off between the coverage of potential multichannel interactions and the computational cost of the procedure (with the number of iterations limited as above, one could only cover all pairwise interactions). A dropout layer in the model architecture enhances parameter randomization, promoting the generalization of the identified ROIs. Still, using a probabilistic scoring method did not significantly affect the results. Section title: 4.3 Evaluation metrics Educational score: 3.0307774543762207 Domain: biomedical Document type: Study Language: en We evaluated precision, recall, F 1 , and classifier convergence times as suggested in Taha and Hanbury . Our main focus was on F 1 scores and convergence times. Given that the data had varying numbers of samples for each class, we used the weighted micro-averaged F 1 score to account for the class imbalance. The F 1 formula below shows how the final score is calculated, given precision, recall, and F 1 score for each class i . This ensures that each class's contribution is proportional to its prevalence in the data set. Section title: 4.3 Evaluation metrics Educational score: 2.6002535820007324 Domain: biomedical Document type: Other Language: en where TP is the number of true positives, FP is the number of false positives, and FN is the number of false negatives. The weights W i correspond to the number of true samples of class i . Section title: 5.1 Classifier performance comparison Educational score: 1.5811786651611328 Domain: other Document type: Study Language: en As indicated in the Evaluation Metrics subsection, we made final comparisons based on F 1 and convergence time. The combined results can be seen in Figures 4 , 5 , and Table 5 . Section title: 5.1 Classifier performance comparison Educational score: 1.6167291402816772 Domain: biomedical Document type: Study Language: en Based on the results, a few major conclusions can be drawn. Section title: 5.2 Classifiers based on neural networks Educational score: 3.4900710582733154 Domain: biomedical Document type: Study Language: en In our study, we applied CNNs and ResNets with various parameters (e.g., the number of residual blocks). Results on their performance are collected in Figure 6 and Table 6 . We hypothesized that models processing temporal-spatial data can yield better results than using single-time observations. Consequently, we involved two approaches as mentioned above, i.e. as an input to ResNet, we used 1-time point data related to the instantaneous view of the brain state and 6-time points corresponding to the dynamics of the brain during the processing of the tasks. Section title: 5.2 Classifiers based on neural networks Educational score: 4.073151111602783 Domain: biomedical Document type: Study Language: en It can be easily drawn that the networks give more accurate results when data in 6-time point segments are used to train the model. Moreover, the higher number of residual blocks within the middle layer does not imply better network performance, which is evident when results for the encoding phase for the 1-time point and the 6-time point are compared. For the former case (1-time point), the best results are obtained for ResNet32, whereas in the latter case, ResNet14 gives better outcomes. Interestingly, the model outperforms the non-neural classifiers only for ENC2 and ENC3 cases. Adding the resting state to the tasks enhances the accuracy only when four classes are considered, i.e. F 1 for ENC4 (RET4) is lower than ENC5 (RET5). The best 1D CNNs found have performance better than any of the considered ResNets, even though they have a considerably simpler architecture. Interestingly, for the encoding phase, they perform comparably or better than any non-neural classifier, but are comparable or worse for the retrieval phase. Section title: 5.3 Explainability: ROI importance Educational score: 4.030542373657227 Domain: biomedical Document type: Study Language: en The ROI pruning procedure, Algorithm 1 , allowed us to see whether, after removing a highly significant feature r * , some other remaining ROIs considerably changed their scores. Such changes are visualized in the upper panel of Figure 7 and in Figure 11 , where in each row of the heatmap green (red) pixels indicate the regions whose importance increased (decreased) due to the removal of a single ROI (cyan tips of blue vertical lines). These changes can be interpreted as the model compensating for the information loss due to the r * removal by using similar information obtained from the several remaining ROIs, whose importance increased. We observed those remaining features improving their importance scores, with just a small overall F 1 performance decrease. The importance of the other remaining ROIs would decrease if the information they carried was only useful in combination with the one that had been removed. Section title: 5.3 Explainability: ROI importance Educational score: 4.021338939666748 Domain: biomedical Document type: Study Language: en Notable examples of correlated clusters of highly important ROIs are the ones responsible for visuals: 53, 54, and 55 (left inferior occipital gyrus, right inferior occipital, and left fusiform gyrus) or 48 and 56 (right lingual gyrus and right fusiform gyrus) appearing in encoding and retrieval experiments without resting state; see Figure 7 . Similarly, certain basal ganglia (39, 40, 71, 72) and some cerebellar areas (97, 103, 105, 108) had correlated scores and were important for the classifier's decisions in encoding experiments against the resting state. The full list of ROI names can be found in Supplementary material . Figure 8 , together with Supplementary Figures 2 – 4 , offer a visualization of where the most important brain regions are located. Section title: 5.3 Explainability: ROI importance Educational score: 4.003911972045898 Domain: biomedical Document type: Study Language: en The ROI importance scores (not corrected for possible correlations) of the tuned LGBM model are shown in Supplementary Figure 1 , together with all the 100 optimisation trials in Supplementary Figures 5 , 6 . Comparing these scores with the ones obtained from pruning, one can notice where the procedure was indeed beneficial. For instance, before pruning the right inferior occipital gyrus (54) had low importance, while the left inferior occipital gyrus (53) was highly important, but after pruning both areas are almost equally highly important. Similarly, before pruning the right middle occipital gyrus (52) was considerably more important than the left middle occipital gyrus (51), but after pruning in most of the experiments they both have medium importance. Section title: 5.3 Explainability: ROI importance Educational score: 3.419964075088501 Domain: biomedical Document type: Study Language: en The respective results obtained from ResNets and Algorithm 2 are shown in Figures 9 , 10 and in Supplementary Figures 9 – 13 . Fewer ROIs than in the previous method reach high scores, notably: 85, 86 visual processing regions, 4 and 6 executive regions, 91 and 92 in the cerebellum, and several auditory ROIs (e.g., 81, 84, 87). Section title: 5.3 Explainability: ROI importance Educational score: 3.258387804031372 Domain: biomedical Document type: Study Language: en Surprisingly, there is no significant correlation between respective ResNet and LGBM importance scores, even though their classification performance is comparable. For 2- and 4-class tasks the encoding and retrieval scores are less correlated for ResNet (correlation coefficient 0.27 and 0.55, respectively) than LGBM (0.94 in both cases), but are similarly correlated for 3- and 5-class tasks (0.48 and 0.57 versus 0.38 and 0.50). The correlation between 2- and 4-class importance scores (0.88 for ENC and 0.76 for RET) and 3- and 5-class (0.89, 0.80) are comparable with LGBM (0.91, 0.92, 0.95, 0.68, respectively). Section title: 6 Discussion Educational score: 4.15507173538208 Domain: biomedical Document type: Study Language: en From the neurocognitive perspective, the discriminant analysis allows us to reveal the dynamic process of working memory. As visualized in Figure 7 , the first 10 steps of pruning showed important ROIs mainly in auditory, sensory-motor, and visual networks in both encoding and retrieval phases. Superior temporal gyrus and temporal pole (ROIs 81, 82, 83) from the auditory network are responsible for the encoding of speech sounds , speech representation , or visual cognition . In the 2-class task, when the distinction between visuospatial and verbal tasks was made, these regions seem to play a significant role . In the visual networks, the important regions are inferior occipital gyri , which are involved in spatial feature processing as well as in insightful problem solving . The results are in line with the sensory reactivation theory, according to which the retrieval phase involves the reinstatement of a process that appeared in the encoding phase . Section title: 6 Discussion Educational score: 4.364252090454102 Domain: biomedical Document type: Study Language: en When we look at the influence of feature pruning on importance scores , it can be easily noticed that all experiments in the encoding phase are very similar, but differences are seen in the retrieval stage. Regarding the results from experiments during encoding, the visual regions (like ROIs 46, 47, 53, 54) gain higher importance, which suggests the differences in the involvement of visual networks related to memorizing different visual stimuli. In the retrieval phase, we also observed the significance of visual brain areas, which could be interpreted using the sensory reactivation hypothesis. However, our results showed mainly the importance of regions in the Visual III network, namely the fusiform gyri and inferior temporal gyri (ITG). The fusiform gyri are engaged in high-level information processing such as object recognition, visual language perception, and visual attention . They are defined as critical structures for visual categorization , whereas the inferior temporal gyri are also involved in visual object recognition, as well as in phonological and lexical processing, and decision-making. ITG is assumed to be a key region in the visual association area with connections from visual regions and high-order areas including the prefrontal cortex . Our analysis revealed the importance of the superior frontal gyrus, a part of the medial prefrontal cortex, which is engaged in higher levels of working memory processing like monitoring and manipulation and in spatial feature processing . The study by Hu et al. showed also the role of the superior frontal gyrus in executive control, more specifically in efficient response inhibition. The results of a recent study using a combination of electrocorticography and direct cortical stimulation suggested that this structure might be a key node coordinating working memory . Our results showing the significance of inferior temporal and superior frontal gyri in retrieval phase seem to confirm the evidence that content representations during encoding and retrieval in WM, differ and engage distinct brain regions. Specifically, the importance of the superior frontal gyri indicates the conversion of content representations from visual to frontoparietal regions . The applying machine learning algorithms allowed us to achieve results that go beyond the sensory reactivation hypothesis. They seem to be in line with the recent evidence showing differences in neural activity patterns of encoding and retrieval processes, revealing change in the neural localization of content representations . Section title: 6 Discussion Educational score: 4.247980117797852 Domain: biomedical Document type: Study Language: en In RET3 and RET5 (the experiments including REST), the regions in basal ganglia and cerebellum networks scored the highest importance. The basal ganglia were shown to be involved in the control of attention to problem features, as well as the transport of information between higher visual regions and the prefrontal cortex. There is also evidence of the basal ganglia-cerebellum-prefrontal cortex network, whose activation is associated with working memory and executive functions . Current computational models shed new light on working memory as a process that strikes a trade-off between stability and flexibility (the core feature of executive control) controlled by the basal ganglia and cerebral cortex . It is well known that dopamine is a key transmitter in the working memory process . The recent study revealed the role of a balance between prefrontal and striatal dopamine secretion and dynamic dopamine-dependent adjustment for adaptive cognition. The results showed the influence of basal ganglia on cognitive control modulation in a way that striatal dopamine controls flexible gating of actions with increasing activity in the direct “go” pathway and decreasing activity of the indirect “no-go” pathway of this structure . Regarding the cerebellum, recent studies have shown that this structure is involved in a much higher number of cognitive functions than was assumed . The basal ganglia and cerebellum regions seem to be key regions in the distinction between cognitive and non-cognitive (REST) tasks, which to our knowledge has been confirmed for the first time in the present neural networks analysis. Section title: 6 Discussion Educational score: 4.148580074310303 Domain: biomedical Document type: Study Language: en Regarding the ResNet model results , high ROI importance was attributed to cerebellum regions and higher-order visual processing areas (Visual III network). Notably, during the retrieval phase, ResNets place importance on the executive network, which is not observed in the LGBM outcomes . This suggests that ResNet may be more effective at identifying structures involved in a task, even though LGBM's importance scores are more consistent. Additionally, when examining the three most important ROIs in the ResNet model , we observe differences in hemispheric locations between the encoding and retrieval phases. These differences seem to support the newly established model of Activity Silent Working Memory, highlighting the involvement of episodic memory in working memory tasks related to context representation . Section title: 6 Discussion Educational score: 4.1657867431640625 Domain: biomedical Document type: Study Language: en The importance scores of the LGBM model, on the other hand, are remarkably similar to the results obtained from measuring temporal correlations in the same data . In particular, the differences in Hurst exponents between the perceptual (GLO, LOC) and verbal similarity (PHO, SEM) tasks pointed to the same regions in Sensory Motor, Visual I and Visual II networks, and partly in Dorsal networks for the encoding phase; a similar pattern in Sensory Motor, Visual I and Visual II networks appeared also in the retrieval phase. There are no such striking similarities with the ResNet results. This observation is intriguing, since the LGBM had no access to the temporal information (classification based on single time points), whereas the Hurst exponents quantify the temporal correlation structure of time series. And vice versa, the Hurst exponents were computed separately for each ROI, whereas LGBM results depend solely on the equal-time cross-correlations between ROIs. Such interconnection between autocorrelation and cross-correlation in fMRI data has been previously observed in Ochab et al. . Section title: 6 Discussion Educational score: 4.115792751312256 Domain: biomedical Document type: Study Language: en Our results based on the new methods of data analysis confirmed the dynamic-processing model of working memory. This process is much more stable in the encoding phase than in the retrieval phase. This stability related to the capacity-limited WM process can be seen in the similarity of the encoding phase, where the differences are revealed only in the visual processing regions regarding specific characteristics of visual stimuli. The retrieval of information from memory is more flexible (changing over time) and is context-dependent. We also observed the change in the neural localization from visual to frontoparietal regions, which is not in line with the sensory reactivation hypothesis. To conclude, we believe that the application of some of the most commonly used machine learning algorithms and neural networks to investigate the encoding and retrieval phases is very promising for future research. | Study | biomedical | en | 0.999999 |
PMC11695340 | Section title: Introduction Educational score: 4.0817790031433105 Domain: biomedical Document type: Review Language: en Coronary artery disease (CAD) is a form of ischemic cardiomyopathy in which the development of atherosclerosis leads to coronary stenosis. In the Asian population, which accounts for nearly half of the world's population, the prevalence of CAD has increased dramatically with the changes in dietary structure resulting from rapid economic development ( 1 ). Although men have a greater risk of developing coronary heart disease overall, CAD remains the leading cause of death among women ( 2 ). Moreover, the protective effect of estrogen is a main factor contributing to the reduced risk of coronary heart disease of women compared with men ( 3 ), and after menopause, the incidence of coronary heart disease in women is comparable to that in men ( 4 ). Women with CAD have a worse prognosis than men with CAD, primarily because women are more likely to experience non-severely obstructive CAD and atypical symptoms ( 5 – 8 ). Accordingly, reliable markers for predicting poor prognosis among post-menopausal women with CAD are urgently needed. Section title: Introduction Educational score: 4.210662841796875 Domain: biomedical Document type: Study Language: en Atherosclerosis encompasses both localized and systemic chronic inflammation triggered by lipid accumulation. Localized inflammation induces the migration and aggregation of peripheral blood immune cells into the subendothelium through activation of vascular endothelial cells and their high expression of chemotactic adhesion molecules ( 9 ). The classical inflammatory marker C reactive protein (CRP) was shown to have independent predictive value in male and female patients with CAD, patients with acute coronary syndrome (ACS) and patients with chronic coronary syndrome (CCS) ( 10 – 12 ). The peripheral blood leukocyte level also has been shown to be strongly and independently associated with CAD, and leukocyte count has been correlated with the diagnosis and severity of CAD as well as with poor prognosis in patients with different types of CAD ( 13 ). Specific correlations have been reported between elevated eosinophil, monocyte, and neutrophil counts among peripheral blood leukocyte counts and an increased risk of CAD ( 14 ). A novel derived inflammatory marker termed the Systemic Inflammatory Response Index (SIRI) has been established as a comprehensive inflammatory indicator that reflects the relative levels and balance of monocytes, neutrophils, and lymphocytes. SIRI was first defined to predict cancer prognosis ( 15 ), and further studies have demonstrated its good predictive value for prognosis in patients with coronary heart disease ( 16 , 17 ). However, to our knowledge, no studies have explored the predictive value of the SIRI for the prognosis of postmenopausal women with CAD. Section title: Introduction Educational score: 4.0502777099609375 Domain: biomedical Document type: Study Language: en The present study aimed to investigate the predictive value of the SIRI for the occurrence of death and adverse cardiovascular and cerebrovascular events in older postmenopausal women with CAD. The results of this study may provide an experimental basis for the clinical application of the SIRI as a valuable biomarker in these patients. Section title: Experimental design and study population Educational score: 4.118488311767578 Domain: biomedical Document type: Study Language: en This study was a single-center, retrospective, observational cohort study and included 672 older postmenopausal female patients who were diagnosed with CAD by coronary angiography at the First Affiliated Hospital of Zhengzhou University from January 2019 to December 2020. Coronary angiography and qualitative and quantitative coronary arteriographic analyses were performed by experienced interventional cardiologists. Patients with no contraindications were treated with oral antiplatelet agents and statins after diagnosis. A complete clinical history was available for all included patients. Section title: Experimental design and study population Educational score: 4.157076358795166 Domain: biomedical Document type: Study Language: en The inclusion criteria for this study were: (1) age ≥50 years, (2) female sex and postmenopausal status; (3) confirmed diagnosis of CAD based on coronary angiography showing stenosis ≥50% in at least one vessel; and (4) complete clinical profile with no missing data. ACS included all cases of unstable angina, ST-elevation myocardial infarction, and non-ST-segment elevation myocardial infarction, whereas CCS was defined as stable angina. The exclusion criteria for this study included the presence of: (1) any acute infectious disease, (2) any tumor type (unless the tumor had been surgically removed or cured), (3) any autoimmune or hematologic disease, and (4) severe heart failure, heart valve disease, structural heart disease, or cardiomyopathy. Section title: Experimental design and study population Educational score: 1.8018988370895386 Domain: biomedical Document type: Study Language: en The study design and procedures complied with the tenets of the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Follow-up data were obtained by reviewing medical records or via telephone interviews. Section title: Definition of SIRI Educational score: 3.8249995708465576 Domain: biomedical Document type: Study Language: en The novel derived inflammatory index SIRI selected for evaluation in this article is an important indicator of the state of systemic inflammation ( 15 ). The SIRI is calculated by multiplying the absolute neutrophil value by the absolute monocyte value divided by the absolute lymphocyte value. Section title: Clinical testing Educational score: 4.126183986663818 Domain: biomedical Document type: Study Language: en Venous blood samples from patients who had been fasting for at least 8 h were collected within 24 h of admission for laboratory testing. Medical history data for each patient, including age, history of diabetes, history of hypertension, and history of atrial fibrillation, were obtained from admission charts. Laboratory results were recorded, including those of routine blood tests (absolute leukocyte count, absolute neutrophil count, absolute monocyte count, and platelet count); lipid levels (total cholesterol, triglycerides, low-density lipoprotein cholesterol [LDL-C], and high-density lipoprotein cholesterol [HDL-C]); fasting glucose and glycosylated hemoglobin; hepatic and renal functional parameters [creatinine, urea, uric acid, alanine transaminase (ALT), aspartate transaminase (AST), total bilirubin, direct bilirubin, indirect bilirubin, and albumin]; and blood coagulation markers (fibrinogen and D-dimer). The number of coronary artery lesions, lesion severity, and the presence or absence of left main artery lesions were recorded according to the patient's coronary angiography findings. Section title: Clinical endpoints and follow-up Educational score: 4.107408046722412 Domain: biomedical Document type: Study Language: en We analyzed both primary and secondary endpoints. The primary endpoint was long-term mortality, including all-cause mortality (ACM) and cardiac mortality (CM). The secondary endpoints included major adverse cardiac events (MACEs) and major adverse cardiovascular and cerebrovascular events (MACCEs). ACM was defined as patient death from any cause, and CM was defined as patient death from a cardiac cause, including heart failure, myocardial infarction, or malignant arrhythmia, and other unexplained deaths for which non-cardiac causes could be definitively excluded. MACEs included ACM, nonfatal myocardial infarction, acute heart failure, target vessel revascularization (percutaneous coronary intervention or coronary artery bypass graft therapy), new malignant arrhythmia on discharge (transient or continuous ventricular tachycardia, ventricular fibrillation, atrial fibrillation, atrial flutter, and second- to third-degree atrioventricular block), and angina pectoris that required hospitalization. MACCEs included MACEs along with cerebrovascular disease, including cerebral hemorrhage and acute cerebral infarction. All patients were followed up for an average of 42 months through follow-up records from telephone calls or visits to the First Affiliated Hospital of Zhengzhou University. Section title: Statistical analysis Educational score: 3.9893059730529785 Domain: biomedical Document type: Study Language: en All data analyses were performed using the statistical software SPSS 26.0 for Windows (SPSS, Inc., Chicago, IL, USA). Continuous data are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was employed to detect differences between groups when continuous variables exhibited both a normal distribution and a chi-square value, whereas the Kruskal–Wallis test was utilized when either of these conditions was not met. Categorical data are expressed as frequencies and percentages, and these variables were compared using the χ 2 test. Kaplan–Meier analysis was applied to compare the cumulative incidence of long-term outcomes between groups. and the log-rank test was used to identify significant differences between groups. To construct Cox models, univariable models were used to identify possible predictive variables. Then variables that were significant ( p < 0.05) in the univariable Cox model were entered simultaneously into the multivariable Cox model. Hazard ratios (HRs) and their 95% confidence intervals (CIs) were calculated. A two-sided p < 0.05 represented a significant difference. Section title: Baseline characteristics of postmenopausal CAD patients in the three SIRI groups Educational score: 3.5561766624450684 Domain: biomedical Document type: Study Language: en Initially, 672 older postmenopausal female patients with a diagnosis of CAD were reviewed, including 327 cases of ACS and 340 cases of CCS. Fifty-five patients were lost to follow-up because their contact information changed during the follow-up period, and these patients were excluded from this study. Finally, a total of 617 patients were included, including 297 cases of ACS and 320 cases of CCS. The patient flow chart is shown in Figure 1 . Section title: Baseline characteristics of postmenopausal CAD patients in the three SIRI groups Educational score: 4.078655242919922 Domain: biomedical Document type: Study Language: en The 617 patients included in this study were divided into three groups according to SIRI tertiles, including the low level group (SIRI <0.73; n = 206), the intermediate level group (1.29≥ SIRI <0.73; n = 205), and the high level group (SIRI ≥1.29; n = 206). The baseline data for the patients in these three groups are presented in Table 1 . The three groups showed statistically significant differences in age, white blood cell count, neutrophil count, monocyte count, lymphocyte count, platelet count, total triglycerides, HDL-C level, fibrinogen level, creatinine level, AST level, albumin level, fasting glucose level, glycosylated hemoglobin level, prevalence of diabetes mellitus, and prevalence of single-branch coronary artery lesions. No significant differences were found among the groups in the prevalence of hypertension, total cholesterol level, LDL-C level, or medication use. None of the patients had a history of smoking or alcohol consumption. Section title: Comparison of the incidence of each outcome among the SIRI groups Educational score: 4.140513896942139 Domain: biomedical Document type: Study Language: en Table 2 presents the comparison of the incidence of each outcome among the three SIRI groups. With 7 (3.4%) ACMs in the low level SIRI group and 19 (9.4%) ACMs in the high level SIRI group, the frequency of ACMs was significantly higher in the high level SIRI group ( p = 0.014). The incidence of CM also was significantly higher in the high level SIRI group than in the low SIRI group (8.4% vs. 2.4%, p = 0.003). Furthermore, both MACEs (41.9% vs. 27.8%, p = 0.01) and MACCEs (45.3% vs. 32.2%, p = 0.024)occurred at a significantly higher frequency in the high level SIRI group compared with the low SIRI group. Section title: Univariable and multivariable Cox regression analyses of predictive factors for each outcome Educational score: 4.158563613891602 Domain: biomedical Document type: Study Language: en To identify independent predictors of each outcome in older menopausal women with CAD, we conducted Cox proportional hazard analyses. The results are shown in Table 3 . All potential confounding variables were initially incorporated into a univariate Cox analysis. Subsequently, significant variables ( p < 0.05) were included in multivariable Cox regression modeling. From the univariable analysis, the incidence rates of ACM, CM, MACEs, and MACCEs were significantly higher in the high level SIRI group than in the low level SIRI group [ACM: HR = 3.214 (95% CI: 1.350–7.649), p = 0.008; CM: HR = 3.979 (95% CI: 1.647–10.792), p = 0.007; MACE: HR = 1.752 (95% CI: 1.252–2.451), p = 0.001; MACCE: HR = 1.650 (95% CI: 1.203–2.264), p = 0.002]. From the multivariable Cox regression analysis, compared with patients in the low level SIRI group, those in the high level SIRI group had a 2.249-fold greater risk of developing ACM (HR = 2.581, 95% CI:1.045–6.373, p = 0.040), a 2.297-fold greater risk of developing CM (HR = 2.798, 95% CI: 0.972–8.060, p = 0.057), a 0.672-fold greater risk of developing MACEs (HR = 1.623, 95% CI: 1.123–2.346, p = 0.01), and a 0.585-fold greater risk of developing MACCEs (HR = 1.558, 95% CI: 1.100–2.207, p = 0.012). Section title: Kaplan–Meier survival analysis Educational score: 4.122302055358887 Domain: biomedical Document type: Study Language: en The Kaplan–Meier survival analysis revealed that patients in the high level SIRI group had greater risks of CM (log rank, p = 0.005), ACM (log rank, p = 0.001), MACEs (log rank, p = 0.003), and MACCEs (log rank, p = 0.005) than those in the low level SIRI group. The results are shown in Figure 2 . Section title: Discussion Educational score: 4.138680458068848 Domain: biomedical Document type: Study Language: en The present study investigated the predictive value of the novel inflammatory marker SIRI for adverse prognoses in postmenopausal women with CAD aged 50 years or older. The results showed that the SIRI could significantly predict several adverse outcomes in this population. In particular, the SIRI had a high predictive value for the risk of death. Cox regression modeling showed that the risk of ACM was 1.581-fold higher in the high level (third tertile) SIRI group compared with the low level (first tertile) SIRI group (HR = 2.581, 95% CI:1.045–6.373, p = 0.040), and the risk of cardiogenic mortality was 1.798-fold higher in the high level SIRI group compared with the low level SIRI group (HR = 2.798, 95% CI: 0.972–8.060, p = 0.057). In addition, the risks of MACEs and MACCEs were 62.3% higher (HR = 1.623, 95% CI: 1.123–2.346, p = 0.01) and 55.8% higher (HR = 1.558, 95% CI: 1.100–2.207, p = 0.012), respectively, in the high level SIRI group compared with the low level SIRI group. The Kaplan–Meier survival curves also showed a gradually shorter survival time and a higher risk of adverse cardiovascular events with increasing SIRI values. Section title: Discussion Educational score: 4.380549430847168 Domain: biomedical Document type: Study Language: en In females, estrogen is known to have anti-inflammatory effects, via downregulation of nuclear factor kappa B (NF- κ B), interleukin-1 (IL-1), and IL-6 and the promotion of anti-inflammatory M2 macrophages, T regulatory cells (Tregs), IL-4 expression, IL-10 expression, and transforming growth factor beta (TGF- β ) expression. Estrogen also contributes to vasodilatation of the vascular endothelium, blockade of hyperglycemia-induced smooth muscle cell proliferation, and promotion of endothelial repair through estrogen receptor alpha (ER α ). Through these combined effects, estrogen has a cardiovascular protective effect in women ( 18 , 19 ). In postmenopausal women after the age of 50 years, the decline in estrogen leads to increases in innate immune activation and pro-inflammatory cytokines, while aging also results in a state of chronic low-grade inflammation, with increased levels of inflammatory cytokines, such as IL-6 and tumor necrosis factor alpha (TNF-α) ( 20 , 21 ). Accordingly, the risk of atherosclerosis is 3.4-fold higher in postmenopausal women than in premenopausal women ( 22 ). In the pathogenesis of ACS, whereas premenopausal women tend to exhibit thick fibrous caps and small necrotic core plaques that undergo erosion, postmenopausal women are more prone to experience rupture of large necrotic core plaques with thin fibrous caps ( 23 ). Histologic studies have shown that plaque rupture is highly correlated with inflammation, whereas no increase in inflammation is found during plaque erosion ( 24 ). The level of CRP, the classic inflammatory factor in peripheral blood, is significantly elevated in postmenopausal women ( 25 ), and a high level of CRP is associated with the occurrence of cardiovascular events (coronary heart disease-related death, nonfatal myocardial infarction or stroke, and coronary revascularization surgery) in postmenopausal women ( 12 ). Elevated CRP levels have also been observed in patients with depression, which is another a high risk factor for coronary heart disease in women ( 26 ). In summary, elevated levels of systemic inflammation in postmenopausal women are associated with the development of CAD and adverse cardiovascular events. The peripheral blood leukocyte level offers a convenient, inexpensive, and readily available indicator of inflammation. Leukocyte counts and neutrophil counts have been shown to be associated with cardiovascular events in postmenopausal women ( 27 , 28 ). However, studies investigating the predictive value of derived inflammatory markers for the prognosis of postmenopausal women with CAD are lacking. In the present study, we demonstrate the good predictive value of SIRI for prognosis in postmenopausal women with CAD. Section title: Discussion Educational score: 4.697573661804199 Domain: biomedical Document type: Study Language: en CAD is a chronic inflammatory disease, and a variety of inflammatory immune cells in the peripheral blood are involved in the formation of atherosclerotic plaques ( 29 ). The SIRI represents the combined level and interbalance of monocytes, neutrophils, and lymphocytes. Mononuclear macrophages are well established as the major immune cells in atherogenesis and are involved in the entire disease process, including the onset and progression of atherosclerosis, formation of unstable plaques, plaque rupture, intra-plaque vascularization, and post-infarction myocardial remodeling ( 30 ). Monocytes adhere and migrate to the subendothelium of arteries through chemokine attraction (mainly CCL2) during the stimulation of inflammation. Once there, they further differentiate into macrophages and then phagocytose lipids to form foam cells, which activate NF- κ B targets to sustain the inflammatory response and further promote the formation of large numbers of foam cells to form the lipid core of plaques ( 31 , 32 ). The monocyte count was shown to predict new development of atherosclerotic plaques and to be an independent predictor of prognosis in CAD ( 33 , 34 ). Neutrophils act as fast reactive inflammatory cells. A previous study showed that blocking the CXCL12/CXCR4 chemokine receptor axis in mice can accelerate the process of atherosclerosis by causing an increase in the number of neutrophils in peripheral blood and plaques ( 35 ). Due to the short half-life of neutrophils in peripheral blood, the role of neutrophils in human atherosclerotic formation needs to be determined in further studies. However, clinical studies have shown that neutrophil counts correlate with the severity of CAD as well as the prognosis of patients with ACS and patients after percutaneous coronary intervention (PCI) ( 36 , 37 ). Different lymphocyte subsets have different functions in atherosclerosis, with T helper 1 (Th1) cells having pro-atherogenic effects and Th2 cells, Tregs and B cells having anti-atherogenic effects ( 38 ). Overall, lymphocytes are protective and regulatory factors in atherosclerosis. In clinical studies, lymphocyte counts have been shown to correlate with poor prognosis in patients with CAD in absolute terms and to have diagnostic value in acute myocardial infarction (AMI) ( 39 ). Section title: Discussion Educational score: 4.252257347106934 Domain: biomedical Document type: Study Language: en Peripheral blood immune cells do not exist independently, and CAD progression requires the participation of all types of immune cells and their combined effects. The novel inflammatory marker SIRI was originally developed for cancer risk prediction ( 40 ). However, this marker has also been widely used for risk prediction in CAD. Two large population-based prospective cohort studies have shown that an elevated SIRI correlates with an increased incidence of all-cause cardiovascular death, cardiac death, stroke (hemorrhagic and ischemic), and AMI ( 17 , 41 ). A single-center prospective study evaluated the predictive value of the MLR, NLR, PLR, SII, and SIRI for the occurrence of MACEs (nonfatal myocardial infarction, nonfatal ischemic stroke, and all-cause death) in patients undergoing PCI for ACS. Although all inflammatory markers were found to correlate with the occurrence of MACEs, the predictive value of the SIRI was superior to that of the other markers ( 42 ). The SIRI has a higher predictive value than SII in predicting the occurrence of postoperative adverse cardiovascular events in patients with AMI (area under the curve = 0.678 for SII and 0.707 for SIRI in the predictive value for MACE) ( 43 ). The SIRI also has predictive value for poor prognosis in non-ST-segment elevation myocardial infarction ( 44 ). A study assessing the predictive value of the novel inflammatory markers SII, SIRI, and AISI for mortality after noncorporeal coronary artery bypass grafting in patients with CAD found that only the SIRI had a good predictive value after correction by multifactorial regression analysis ( 45 ). The SIRI has a higher sensitivity for risk prediction of the occurrence of adverse events related to CAD than other derived inflammatory indicators. In conclusion, a multitude of studies have substantiated that SIRI is linked to the incidence of adverse cardiovascular events in patients with varying degrees of CAD. Nevertheless, no study had yet investigated the predictive value of SIRI for the prognosis of patients with CAD in different age and gender subgroups. In the present study, other derived inflammatory indicators, including the MLR, NLR, PLR, and SII, did not have good predictive value for poor prognosis of CAD in postmenopausal women aged 50 years and older, whereas the SIRI did. A recent study indicate that elevated SIRI values are significantly associated with an increased risk of stroke and its subtypes in elderly patients with hypertension ( 46 ). For postmenopausal women as a specific study population, recent studies have found that an elevated SIRI is associated with cardiovascular death in postmenopausal women with osteoporosis or reduced bone mass ( 47 , 48 ). This may be due to the involvement of multiple immune cells and inflammatory mediators in the pathogenesis of osteoporosis and atherosclerosis ( 49 ). In a study of the association between derived inflammatory indicators and the occurrence of ACS in 250 elderly women, the SIRI was found to be significantly higher in patients with ACS but was not included in the construction of logistic regression models after elimination of covariates by backward stepwise regression analysis ( 50 ). It is evident that a correlation exists between the SIRI and a wide range of diseases affecting the cardiovascular system, as well as other systemic diseases. Furthermore, the SIRI is an excellent indicator of the level of systemic inflammation. Section title: Discussion Educational score: 4.099137306213379 Domain: biomedical Document type: Study Language: en The present study demonstrates for the first time that the novel derived inflammatory index SIRI has good predictive value for the assessment of multiple adverse outcomes in postmenopausal women with CAD. Thus, the SIRI is anticipated to serve as a pivotal reference index and scoring program for prognostic assessment of clinical postmenopausal women with coronary artery disease. Some limitations of the study should be noted. First, this study was a single-center retrospective cohort study with limited sample size, and thus, the results need to be further validated in a multicenter study with a large sample set and, ultimately, by meta-analysis in the future. Furthermore, despite our efforts to mitigate confounding factors through a sufficient sample size and rigorous statistical techniques, retrospective studies are inherently constrained by limitations such as selection bias, information bias, recall bias, and forgetting bias. Also, in this study, patients were not further divided into subgroups according to different clinical conditions/comorbidities to analyze the prognostic value of SIRI. In the future, a larger sample size is needed to explore the predictive value of SIRI for the prognosis of postmenopausal patients with ACS, CCS, and those suffering from comorbidities. Section title: Conclusion Educational score: 4.0658721923828125 Domain: biomedical Document type: Study Language: en In conclusion, our study demonstrates that SIRI is an independent predictor of poor prognosis in postmenopausal women with CAD, including CM, ACM, MACEs, and MACCEs. Accordingly, the SIRI can be used in strategies to identify high-risk groups among postmenopausal women with CAD. | Review | biomedical | en | 0.999998 |
PMC11695344 | Section title: Introduction Educational score: 4.472848892211914 Domain: biomedical Document type: Study Language: en Muscle-invasive bladder cancer (MIBC) is a malignancy of the urinary system, which is characterized by a high degree of heterogeneity, recurrence, and metastasis, leading to a poorer prognosis ( 1 ). The tumor microenvironment (TME) is considered an important determinant in cancer biology, tumor progression, and therapeutic responses ( 2 ). Previous studies have reported that the interactions between tumor cells and TME are crucial for tumor development and a significant driving force in tumor deterioration ( 3 , 4 ). Fibroblasts (FB) represent an important component of TME. The stroma of normal bladder tissues contains quiescent or resting FB ( 5 , 6 ). FB is activated during neoplastic cell invasion for the reconstruction of the surrounding environment ( 7 ). Then, the invaded cancer cells interact with novel environments, creating conditions conducive to the survival and migration of tumor cells ( 8 ). Cancer cells interact with neighboring cells and non-cellular components in the TME, influencing tumor growth, invasion, metastasis, and the efficacy of treatment ( 8 ). TME-related targeted therapies have recently garnered increasing attention ( 2 ). Nevertheless, the underlying reason for poorer prognosis in bladder cancer patients following the invasion of tumor cells into the muscular layers, and whether this is associated with the TME, remains unknown. Hence, investigating the spatial characteristics of the MIBC microenvironment and the spatial interactions between cells may reveal important factors driving tumor progression. Section title: Introduction Educational score: 4.098783493041992 Domain: biomedical Document type: Study Language: en Recent advancements in spatial omics offer a new perspective, allowing the analysis of in-situ expression profiles and spatial distributions of TME components ( 3 , 9 ). By describing cellular interactions and neighborhood patterns based on a spatial perspective, the communication networks within the TME were discovered ( 10 ). These analyses facilitate the identification of biomarkers, modulation of anti-tumor immunity, and formulation of targeted therapies for different cancers including colorectal ( 11 ), breast ( 12 ), bladder ( 13 ), and lung cancer ( 14 ). Imaging mass cytometry (IMC) has become an important tool in TME study ( 15 ). IMC allows the concurrent identification of numerous protein expressions in tissue sections, providing critical spatial data that allows for the detailed characterization of the TME’s complex spatial architecture ( 16 ). Section title: Introduction Educational score: 4.273507595062256 Domain: biomedical Document type: Study Language: en In this study, we collected 40 MIBC tissue sections. IMC was used to determine the protein expression of 185 regions of interest (ROIs), which included 100 tumor regions (T), 62 leading-edge regions (L), and 23 nontumor regions (N). We found that the spatial structural characteristics changed from N to L to T regions. Through the in-depth analysis of the components in N, L, and T regions, four different FB clusters with unique phenotypes were found. Each of the FB clusters showed specific spatial distribution patterns. Notably, we found that a specific FB cluster (S3) is associated with abnormal angiogenesis and poor prognosis. Single-cell RNA-seq (scRNA-seq) and spatial transcriptome (ST) analyses verified that S3 interacted with endothelial cells via the receptor–ligand (L–R) pair, NOTCH1–JAG2. Lastly, based on the molecular characteristics of S3, we identified an S3-related prognostic signature, which can provide prognostic insights into patients with cancers. Altogether, the findings of this study provide novel insights into the spatial diversity from N to L to T regions, offering evidence to determine the diagnostic and therapeutic targets of MIBC. Section title: Patients and samples Educational score: 3.912395477294922 Domain: biomedical Document type: Study Language: en Forty paraffin histopathological sections were obtained from the pathology department of the First Affiliated Hospital of Guangxi Medical University. All participants had not received any tumor therapy before enrolment, and the diagnosis of MIBC was confirmed by two experienced pathologists. The N, L and T region were delineated by the pathologist. Written informed consent was provided by all participants. This study was approved by the ethics committee of the First Affiliated Hospital of Guangxi Medical University . The following cohort were used for pan-cancers analysis: TCGA-BLCA, TCGA- MESO, TCGA- GBM, TCGA-LGG, TCGA-STAD, TCGA-UVM, TCGA-KIRC, TCGA-KIRP, TCGA-LUSC, GSE32894, GSE188715. The bulk RNA-seq data and clinical information of TCGA datasets can be downloaded from https://portal.gdc.cancer.gov/ . As for GEO datasets, they can be found at https://www.ncbi.nlm.nih.gov/geo/ . Section title: Imaging mass cytometry analysis Educational score: 4.09149169921875 Domain: biomedical Document type: Study Language: en IMC was performed as previously described ( 17 ). Briefly, after dewaxing the tissue sections (thickness of 5µm), antigen retrieval was performed using Tris-EDTA antigen retrieval solution (FC16FA0005, Sangon Biotech (Shanghai) Co.,Ltd.,China). The sections were then blocked with a 3% BSA solution at room temperature and incubated overnight at 4°C with antibody cocktail, as shown in Supplementary Table S1 . The next day, the sections were stained with Cell-IDTM Intercalator-Ir (Fluidigm) for DNA and subjected to analysis. Section title: Imaging mass cytometry analysis Educational score: 4.154253959655762 Domain: biomedical Document type: Study Language: en The MCD viewer software was used to open the files generated by the IMC system, and 16-bit OME-TIFF files were exported. The 16-bit OME-TIFF files were imported into the CellProfiler software, where channel information was set and signal intensities of each channel were extracted. The DNA (Ir191 and Ir193) and cell surface markers were used to identify the cell nuclei and cell boundaries, respectively. Single cells were then segmented and TIFF files were exported. The TIFF files exported from CellProfiler were imported into the HistoCAT software. The phenograph algorithm was applied for clustering, while the tSNE algorithm was used for high-dimensional data reduction. Neighbor analysis was employed to examine the interactions between different cell clusters. The immune cells and stromal cells were gated in cytobank ( https://community.cytobank.org ) by specific markers: stromal cell (Collagen I, vimentin), immune cell (CD45), B cell (CD20), macrophage (CD68), CD8+ T cell (CD3, CD8) and CD4+ T cell (CD3, CD4). Section title: Imaging mass cytometry analysis Educational score: 4.1219072341918945 Domain: biomedical Document type: Study Language: en During cell division, the X/Y coordinates of each cell type were determined. Similarly to previously reported methods ( 18 ), we utilize the euclidean distance to calculate the closest distance between each cell and different cell types. On a per-ROI basis, we then computed the average closest distance between cell types within each ROI. Cellular neighborhood identification: For the 1,303,975 cells in our experiment, we set the window size to 26, consisting of one central cell and its 25 nearest neighbors, determined by euclidean distance in X/Y coordinates. We chose a 25-cell radius as a rough approximation, where the central cell distance in each direction is visually determined to be a good indicator of local functional activity. Section title: Single-cell RNA sequencing analysis Educational score: 4.113454818725586 Domain: biomedical Document type: Study Language: en The scRNA-seq data from eight bladder cancer patients were downloaded from the cited literature ( 19 ). As a quality control measure, we excluded sequenced cells that had less than 200 detected genes and more than 10% mitochondrial genes. Additionally, genes expressed in fewer than three cells were also excluded. We used the FindClusters function in Seurat (version: 4.0.1) to identify 35 clusters and visualize them using a t-distributed stochastic neighbor embedding (t-SNE) plot. Cluster-specific genes were used to annotate cell types with classic markers described in previous studies ( 19 ): tumor epithelial cells (EPCAM+/KRT17+); endothelial cells (PECAM1+/ENG+); fibroblasts (COL1A1+/RGS5+); myeloid cells (LYZ+/CD68+); T cells (CD3D+/CD2+); B cells (CD79A+/MZB1+); and mast cells (TPSAB1+/CPA3+).The “ComplexHeatmap” R package was used to visualize the average expression values of marker genes for each cell cluster. Section title: Single-cell RNA sequencing analysis Educational score: 4.0747785568237305 Domain: biomedical Document type: Study Language: en For the analysis of interactions between cell types, we extracted the expression profiles of each cell type from eight bladder cancer tissues. The expression matrix, along with cell type metadata, was used as input for running cellphonedb with the statistical analysis method in Python. This study detected statistically significant interactions (p < 0.05) between receptors and ligands by comparing them against a curated L-R database. The “ktplots” R package was used to visualize the significant L-R pairs identified by cellphonedb. Transcription factor prediction of S3 up-regulated gene was performed by BART, an online analytical tool ( http://bartweb.org/ ). Section title: Spatial transcriptome analysis Educational score: 4.091526031494141 Domain: biomedical Document type: Study Language: en Bladder cancer ST data can be obtained from published literature ( 13 ). The “SpaCET” R package was utilized to compute the proportion of tumor cells and other cell types for each spot. Spots with more than 60% tumor cells, FB, or endothelial cells were designated as tumor region, FB region, or endothelial region, respectively ( 20 ). We estimated S3 enrichment in spots by scoring each spot of the ST data using S3 up-regulated genes. Positive L-R spots need to meet the following criteria: 1. They should be located at the boundary between the FB and endothelial region. 2. The ligand and receptor should be queryable in the cellponedb database. 3. The spot should express both the corresponding ligand and receptor genes simultaneously. Section title: Spatial transcriptome analysis Educational score: 2.9755585193634033 Domain: biomedical Document type: Other Language: en The gene sets used for ST scoring can be obtained from the GSEA official website ( https://www.gsea-msigdb.org/gsea/downloads.jsp ). The GSVA package (version: 1.38.2) was employed to calculate the enrichment score for each spot. Section title: Establishment of FCS3S prognostic prediction system Educational score: 4.0924882888793945 Domain: biomedical Document type: Study Language: en Based on the expression of specific markers (Collagen1, CD90, Vimentin, and α-SMA), FB can be classified into FB S3, FB S4, and FB SX. “FindMarkers” was utilized to perform differential gene expression analysis for FB subtypes, with logFC. threshold >= 0.25 and p _val_adj < 0.05 considered as significantly differentially expressed genes. Further filtering of the characteristic genes specific to FB S3 was performed using xgboost. Section title: Statistical analysis Educational score: 3.807563543319702 Domain: biomedical Document type: Study Language: en Statistical analysis was conducted using two-sided Student’s t-test and Kruskal-Wallis rank sum test. A significance level of p <0.05 was considered statistically significant. Kaplan-Meier analysis was used to estimate the overall survival curves of patients with MIBC, and the log-rank test was employed to compare the curves. GraphPad Prism 8 software was utilized for statistical analysis. Section title: Spatial heterogeneity of the tumor microenvironment in MIBC Educational score: 4.139270782470703 Domain: biomedical Document type: Study Language: en To explore the complex spatial characteristics of the MIBC microenvironment and enhance our understanding of its role in promoting metastasis. We performed IMC with a panel of 33 TME-associated markers to detect 100 tumor core regions (referred to as T), 62 leading-edge regions (referred to as L, the boundary of tumor cell invaded into muscular layer), and 23 nontumor regions (referred to as N) from paraffin-embedded tissue sections of 40 patients with MIBC, yielding 6,105 high-quality images . Through cellular identification and segmentation of these images, we obtained single-cell expression profiles of 33 proteins . Segmentation obtained data from 185 ROIs, ranging from 1562 to more than 10,000 cells per ROI . Section title: Spatial heterogeneity of the tumor microenvironment in MIBC Educational score: 4.149148941040039 Domain: biomedical Document type: Study Language: en We found that the spatial structure of N, L, and T regions showed significant heterogeneity . By cell clustering and spatial reconstruction, we found that cells with similar phenotypes cluster nearby within the T and N regions. On the other hand, cells in the L region exhibit a more disorganized distribution, suggesting a more complicated microenvironment in the L region compared with that in the T and N regions . The deep analysis using Cytobank (an online analytic platform) revealed that the frequencies of stromal cells decreased from N to L to T regions progressively. The frequencies of immune cells including B cells, macrophages, CD4+ T cells, and CD8+ T cells increased in the L region than in the N and T regions . Altogether, these results suggest that the microenvironments of the N, L, and T regions are spatially heterogeneous in MIBC. Section title: The spatial characteristics of microenvironmental components exhibit regional diversity Educational score: 4.259500980377197 Domain: biomedical Document type: Study Language: en Subsequent analyses were performed to determine the spatial heterogeneity of the microenvironment in N, L, and T regions. In the N region, numbers of B cells were recruited, whereas T cells and macrophages were less prevalent. The L regions displayed a higher density of both B cells and T cells, which were positioned near each other, with macrophages more dispersed near the tumor cells. The T regions showed a decreased number of immune cells, which were primarily located in the stromal areas rather than infiltrating the cancer nests . The collagen structures in these regions also exhibited regional heterogeneity. The L regions were characterized by disordered collagen configurations, and some collagen appeared as dots . Collagen was observed as curved structures in the N regions, loosely encasing small segments of muscle tissue . Within the T regions, the following four unique types of collagen structures were identified: curved collagen encapsulating the tumor nests, parallel collagen aligning along the cancer nest margins, directionally arranged collagen, and disorderly arranged collagen with unclear boundaries relative to the tumor . Additionally, tumor budding cells were found in the invasive region, which were dispersed within the tumor stroma as single cells or small clusters . Section title: Specific fibroblast cluster serves as a significant prognostic factor of MIBC Educational score: 4.152197360992432 Domain: biomedical Document type: Study Language: en To explore the important components of the TME involved in the recurrence and metastasis of MIBC, we clustered cells from 100 T regions, 62 L regions, and 23 N regions into 100 cell clusters with distinct phenotypes . Then, we consolidated these 100 clusters into 17 groups based on the expression of specific markers for tumor cells, immune cells, stromal cells, vascular endothelial cells, and muscle cells. Among these were the following four unique FB clusters: FB S1 (α-SMA− Collagen I+ Vimentin- CD90+), FB S2 (α-SMA+ Collagen I+ Vimentin- CD90+), FB S3 (α-SMA+ Collagen I+ Vimentin+ CD90+), and FB S4 (α-SMA- Collagen I+ Vimentin+ CD90+) . These FB clusters exhibited unique spatial distributions. The L regions predominantly contained two types of FB, with nearly 75% being FB S3, referred to as S3 hereafter. Furthermore, all four FB clusters were present in the T regions, whereas the N regions exclusively contained FB S4 . Section title: Specific fibroblast cluster serves as a significant prognostic factor of MIBC Educational score: 4.394146919250488 Domain: biomedical Document type: Study Language: en The L regions, a critical boundary for tumor invasion and metastasis, emerged as the most dynamic area, with S3 as the predominant FB type. This suggests that S3 exhibits high activity and functional characteristics, facilitating tumor progression. Survival analysis showed that MIBC patients with S3-positive tumors had shorter overall survival than those with S3-negative tumors , indicating a crucial role of S3 in MIBC progression. Investigating the TME heterogeneity between S3-positive and S3-negative tumors, we found that S3-positive tumors exhibited a higher density of blood vessels, which included more extensive and severely distorted blood vessels with abnormal dilation. Furthermore, these tumors showed high expression of VEGFR on vessel walls and outward expansion , suggesting that S3 may contribute to abnormal angiogenesis. Subsequent investigation using IMC spatial analysis determined the interactions between S3 and endothelial cells. By evaluating spatial coordinates, we determined the distances from each FB cluster to the nearest endothelial cells and found that the proximity of S3 to endothelial cells was shorter compared with other FB types . Neighbor component analysis among the four FB clusters showed that endothelial cells contained 2.38%, 1.43%, 8.39%, and 2.79% of the neighbors for FB S1, S2, S3, and S4, respectively . These findings reveal an important close interaction between S3 and endothelial cells, highlighting the significant effect of S3 on MIBC angiogenesis. Section title: ScRNA-seq analysis reveals the association between fibroblast cluster S3 and angiogenesis Educational score: 4.352024078369141 Domain: biomedical Document type: Study Language: en To elucidate the mechanisms underlying S3, we analyzed publicly available scRNA-seq data from eight bladder cancer samples. Through cell clustering and annotation, we found 35 unique cell clusters characterized by their specific markers, which included B cells, endothelial cells, tumor epithelial cells, fibroblasts, mast cells, myeloid cells, and T cells . Subsequent refinement of our analysis by reclustering the fibroblasts yielded 14 fibroblast clusters . Using the expression of stromal cell-specific markers, including Collagen 1, α-SMA, Vimentin, and CD90, we identified the previously characterized FB clusters S3 and S4 from IMC analysis and labeled the remaining FB clusters as FB SX . Differential expression analysis showed unique molecular profiles among these FB types . The gene expression upregulated in S3 was enriched in pathways related to angiogenesis, cell migration, epithelial cell proliferation and differentiation, endothelial cell proliferation, and fibroblast proliferation . Analysis of cellular communication among clusters via single-cell L–R interaction showed that S3 engaged in more L–R pair interactions with other cells compared to FB S4 and SX. Specifically, S3 exhibited a significantly higher number of L–R pairs with endothelial cells, showing strong communicative activity . These findings indicate that S3 maintains active communication with different cell types and also engages in close interactions with endothelial cells. Section title: The fibroblast S3 interacts with endothelial cells by L–R pair NOTCH1–JAG2 Educational score: 4.167995929718018 Domain: biomedical Document type: Study Language: en To elucidate the mechanisms between S3 and endothelial cells, we analyzed L–R pairs unique to S3 and endothelial cells . While scRNA-seq offers insights into the cell–cell interactions based on L–R gene expression, it does not account for cell localization. Spatial transcriptomics (ST) analysis, which profiles spatial gene expression within tissues, allows the detection of cell–cell interactions across different regions. By analyzing publicly available ST data from bladder cancer, we identified specific regions corresponding to tumor cells, FB, and endothelial cells. Furthermore, the L–R pair NOTCH1–JAG2 was co-located in the endothelial spots adjacent to the FB region, indicating that S3 may predominantly interact with endothelial cells through the NOTCH1–JAG2 pathway . Section title: The fibroblast S3 is potentially regulated by YAP1 Educational score: 4.164333820343018 Domain: biomedical Document type: Study Language: en Further investigation into the regulatory mechanisms of S3, we employed the BART transcription factor prediction tool to analyze transcription factors associated with genes upregulated in S3 relative to other FB clusters. YAP1 emerged as one of the top predicted transcription factors . IMC imaging confirmed high YAP1 expression in the microenvironment where S3 is located, contrasting with lower expressions in microenvironments dominated by FB S1, S2, and S4 , suggesting a potential regulatory link between S3 and YAP1. To further determine the spatial characteristics and mechanisms associated with S3, we analyzed ST data from bladder cancer. ssGSEA results showed that S3-enriched samples showed higher angiogenesis signature scores, consistent with increased YAP1 expression compared with low-S3-enriched samples . Altogether, these findings show that S3 may promote angiogenesis and could be regulated by YAP1. Section title: Construction of an S3-related prognostic signature based on machine learning Educational score: 4.371066570281982 Domain: biomedical Document type: Study Language: en To investigate the relationship between the presence of S3 and poor prognosis in MIBC, we utilized the XGBoost algorithm to analysis the gene expression profiles of S3 in scRNA-seq and construct an S3-related signature ( Supplementary Table S2 ). Using this signature, we calculated the Fibroblast Cluster S3 Score (FCS3S) in the TCGA-BLCA cohort with the gene set variation analysis (GSVA) algorithm. Our results revealed that patients with high FCS3S had significantly poorer overall survival . This finding was supported by two independent BLCA cohorts . Subsequent analysis showed that patients with high FCS3S exhibited increased activation of pathways associated with malignant tumor progression, including angiogenesis, tumor stemness, cellular exosomes, immune suppression, extracellular matrix (ECM) remodeling, neuroendocrine differentiation, and epithelial–mesenchymal transition (EMT) in the TCGA-BLCA cohort, indicating a higher degree of malignancy in these patients . ESTIMATE analysis showed that patients with high FCS3S had increased immune and stromal scores, as well as overall ESTIMATE scores, compared to those with low FCS3S . Notably, the immune suppression score was significantly elevated in high FCS3S patients, suggesting greater immune suppression in this group . Furthermore, we evaluated the prognostic value of FCS3S across various cancers and found that it can effectively identify patients with poor outcomes and the following eight cancer types: glioblastoma multiforme (GBM), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), lung squamous cell carcinoma (LUSC), uveal melanoma (UVM), lower-grade glioma (LGG), stomach adenocarcinoma (STAD), and mesothelioma (MESO) . To summarize, our findings verified that S3 is complicated and related to the progression of MIBC, and FCS3S serves as a robust tool for predicting patient outcomes across different cancer types. Section title: Discussion Educational score: 4.225867748260498 Domain: biomedical Document type: Study Language: en The TME has garnered immense attention in cancer research. Recent advancements in scRNA-seq and cytometry by time-of-flight (CyTOF) have revealed unique phenotypic diversity and heterogeneity within the TME of MIBC ( 17 , 21 ). However, a comprehensive understanding of the spatial landscape of the TME in MIBC has yet to be completely achieved. To address this gap, we performed a spatially resolved analysis that integrated IMC, scRNA-seq, and ST profiling. In the present study, IMC with 33 TME-associated markers was used, producing 6,105 highly multiplexed images from 40 patients. IMC allowed an in-depth analysis of spatial heterogeneity across the N, L, and T regions. We identified four distinct FB clusters in these regions, each with unique phenotypes and spatial distribution patterns. Furthermore, FB S3 (S3) was predominantly enriched in the L regions and closely associated with poor prognosis and angiogenesis. Subsequent investigations using scRNA-seq and ST analyses verified that S3 is complexly involved in angiogenic processes and interacted with endothelial cells via a unique L–R pair, NOTCH1–JAG2. Based on these aforementioned findings, we developed the FCS3S, a prognostic prediction model based on the characteristic genes of S3, which can accurately predict prognostic information in patients. Section title: Discussion Educational score: 4.478826522827148 Domain: biomedical Document type: Study Language: en FB, the primary stromal cells in the TME, are crucial in collagen production ( 22 ). Many scRNA-seq studies have shown the presence of diverse fibroblast subpopulations within the TME, each characterized by novel molecular signatures ( 23 , 24 ). In the present study, we used IMC to identify four distinct FB clusters in the TME of MIBC, each exhibiting different spatial distributions. Four unique types of FB were found in the T regions, whereas only specific types were present in the L and N regions. This heterogeneity indicates that each FB cluster may perform specific biological roles and respond differently to environmental stimuli. Furthermore, S3 emerged as the predominant FB localized in the L region—an area important for studying tumor invasion and metastasis, where tumor cells invade normal tissue and interact with stromal cells ( 25 ). The preferential localization of S3 within this active region indicates that it represents a highly active FB cluster, potentially arising from tumor cells training the resident fibroblasts. Survival analysis showed that patients with S3-positive tumors had significantly shorter overall survival compared with those with S3-negative tumors, highlighting the important role of S3 in the progression of MIBC. IMC analyses showed that relative to S3-negative tumors, S3-positive tumors exhibited higher vascular density, more extensive and morphologically complex blood vessels, and upregulated expression of VEGFR on the vessel walls, indicative of abnormal angiogenesis. Studies suggest that FB can secrete various factors, including VEGFA, PDGFC, FGF2, and osteopontin, which are known to stimulate angiogenesis in tumor tissues ( 26 ). Furthermore, FB facilitates angiogenesis by remodeling the ECM, affecting mechanical properties such as hardness, elasticity, and interstitial fluid pressure of the tumor stroma ( 27 ). They can also recruit endothelial progenitor cells to promote tumor neovascularization by secreting SDF1 ( 28 ). The tissue remodeling images obtained via IMC showed proximity between S3 and endothelial cells, further establishing an association between S3 and angiogenesis. Altogether, these findings suggest that S3 plays an important role in promoting abnormal angiogenesis in tumors. Section title: Discussion Educational score: 4.315662384033203 Domain: biomedical Document type: Study Language: en The emergence of scRNA-seq has revealed the complexity of the TME of MIBC, underscoring the unique phenotypes within its cellular components. To determine the association between S3 and angiogenesis, we analyzed published scRNA-seq data from bladder cancer. Through clustering and cell annotation, we identified S3 at the transcriptomic level, consistent with previous characterization by IMC. Differential analysis showed that genes upregulated in S3 were significantly enriched in angiogenesis pathways compared with those with other FB clusters. L-R analysis showed that S3 exhibited the strongest interactions with endothelial cells, indicating a strong influence on angiogenic processes. To further elucidate the molecular mechanisms underlying the promotion of abnormal angiogenesis by S3, we performed an L–R analysis to determine specific pairs exclusive to S3 and endothelial cells. Nevertheless, this method, based solely on gene expression, lacks spatial context regarding cellular localization. To confirm our findings, we explored publicly available ST data for bladder cancer. Analysis of spatial gene expression patterns within the tissue identified a notable colocalization of NOTCH1–JAG2, a distinct L–R pair between S3 and endothelial cells. Pietras et al. reported that JAG2 can activate the NOTCH signaling pathway under hypoxic conditions to promote endothelial cell tube formation ( 29 ), elucidating a mechanism by which S3 may increase abnormal angiogenesis via the NOTCH1–JAG2 interaction. Section title: Discussion Educational score: 4.216306209564209 Domain: biomedical Document type: Study Language: en Further analysis to elucidate the regulatory mechanisms of S3 involved a transcription factor prediction for genes significantly upregulated in S3. YAP1 was identified as the third-ranked transcription factor. YAP1 is a major mediator in signaling pathways associated with tumorigenesis and exhibits widespread activation in malignant human tumors ( 30 ). Recent a study has reported the close relationship between YAP1 and FB activation, emphasizing the role of YAP1 in influencing FB behavior ( 31 ). Bora–Singhal et al. reported that YAP1 can increase tumor angiogenesis by upregulating VEGFA expression ( 32 ). Our IMC-based tissue remodeling analysis further showed significantly upregulated YAP1 expression within the microenvironment of S3, both in stromal and tumor cells, compared with other FB clusters. Altogether, these findings strongly suggest that S3 may be intricately regulated by YAP1 signaling pathways, contributing to its proangiogenic properties. Section title: Discussion Educational score: 4.28083610534668 Domain: biomedical Document type: Study Language: en To investigate the prognostic role of S3, we used machine learning algorithms to identify the characteristic genes associated with S3. Using the GSVA algorithm, we assigned a prognostic score based on these genes to each patient. Our findings showed that the FCS3S effectively identified patients with unfavorable prognoses across various cancer types, including MIBC, MESO, GBM, LGG, STAD, UVM, KIRC, KIRP, and LUSC. Patients in the TCGA-BLCA cohort with high FCS3S levels exhibited higher stromal and immune scores than those with low FCS3S levels. They also showed upregulated expression of genes related to malignant tumor progression, such as angiogenesis, tumor stemness, and immune suppression. These findings suggest that FCS3S is significantly associated with tumor progression, and patients with high FCS3S levels may respond to antiangiogenic therapies and immunotherapy. Presently, anti-VEGF drugs, including sunitinib, axitinib, and sorafenib are approved for treating advanced and metastatic tumors. However, the development of resistance to anti-VEGF therapy poses considerable challenges ( 33 ). Combining antiangiogenic agents and immune checkpoint inhibitors may have synergistic effects in promoting tumor vessel normalization and stimulating immune activation ( 34 ). Previous studies indicate that tumor vessel normalization can improve the aggregation of immune cells and boost immune function, whereas immune cell activation reciprocally promotes vessel normalization. Their combined application establishes a positive feedback loop, thereby exhibiting potent antitumor effects ( 35 ). Furthermore, in patients with high FCS3S, a promising approach can involve combination therapy using anti-VEGF drugs alongside immunotherapy, leading to more favorable clinical outcomes. Section title: Discussion Educational score: 4.170939922332764 Domain: biomedical Document type: Study Language: en To conclude, we described the spatiotemporal heterogeneity of the microenvironment in MIBC, underscoring the unique spatial structure of the N, L, and T regions, and the enrichment of the specific S3 at the L regions. This FB cluster was closely associated with adverse prognoses in MIBC, playing an important role in promoting aberrant tumor angiogenesis via the modulation of the NOTCH1–JAG2 L–R pair. Based on these insights, we established an S3-associated prognostic signature, which can be effective in the prognostic evaluation of patients with any nine different cancer types. Altogether, our findings provide novel insights into the TME of MIBC, offering novel opportunities and targets for the personalized treatment of MIBC. | Study | biomedical | en | 0.999997 |
PMC11695345 | Section title: Introduction Educational score: 4.15395975112915 Domain: biomedical Document type: Study Language: en Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines administered intramuscularly have played a significant role in protecting against severe coronavirus disease-19 (COVID-19) ( 1 , 2 ). We previously described that the intensity and latency of antibody response to intramuscular SARS-CoV-2 mRNA vaccines were suppressed in patients with muscular disorders (MDs) compared with those in patients without MDs, and we described that MDs may be a key contributor in predicting the antibody response of intramuscular SARS-CoV-2 mRNA vaccines ( 3 ). In a previous study, we found correlations between anti-SARS-CoV-2 spike protein receptor-binding domain (S-RBD) IgG antibody levels and patient age, specifically in patients with Duchenne muscular dystrophy (DMD), which causes progressive muscular degeneration and wasting ( 3 ). We speculated that both the quality and quantity of the muscle might affect the immunogenicity of intramuscular SARS-CoV-2 mRNA vaccination in patients with MDs, and we hypothesized that SARS-CoV-2 immunization in patients with MDs warrants further investigation ( 3 ). Section title: Introduction Educational score: 3.9581031799316406 Domain: biomedical Document type: Review Language: en In 1985, Kawai et al. ( 4 ) described the usefulness of muscle computed tomography (CT) in patients with DMD. Since then, CT as with magnetic resonance imaging (MRI) are considered the gold standard for the noninvasive assessment of muscle quality and quantity and have been used to evaluate patients with MDs in Japan ( 5 , 6 ). Although MRI has been used to quantify muscle volume and composition, thus allowing for the differentiation of muscle tissue from adipose, edematous, and fibrous connective tissues ( 7 , 8 ), CT has the ability to measure muscle quality and quantity, easily and immediately. Therefore, the use of CT for research on muscles is becoming more common ( 8 – 10 ). Section title: Introduction Educational score: 3.9777638912200928 Domain: biomedical Document type: Study Language: en In this study, mean CT values and areas of the deltoid muscle were measured using the CT images of patients with or without MDs who were vaccinated with two doses of intramuscular SARS-CoV-2 mRNA vaccines. We discussed the findings on the association of the immunogenicity of intramuscular SARS-CoV-2 mRNA vaccination with CT muscle images in patients with or without MDs. Section title: Ethical compliance Educational score: 2.9527747631073 Domain: biomedical Document type: Study Language: en This study was approved as an additional study on the immunogenicity of intramuscular SARS-CoV-2 mRNA vaccination in patients with or without MDs by the ethics committee of Nagara Medical Center . This study was assessed using an opt-out consent approach. This means that patients or the parents of patients with intellectual disabilities are included in the retrospective study unless they give their express decision to be excluded. This approach was adopted because of the low risk and potential benefits of the study to patients regarding intramuscular SARS-CoV-2 mRNA vaccination. Section title: Study participants and evaluations Educational score: 4.105100154876709 Domain: biomedical Document type: Study Language: en This study included 75 patients with or without MDs, particularly those with severe motor and intellectual disabilities (SMIDs). Detailed information on the patients and the methods of the previous study were described in a previous article ( 3 ). Patients with or without MDs were vaccinated with two doses of BNT162b2 vaccines (Pfizer-BioNTech). Serum samples were collected from each patient on the day of the second dose of vaccination and then after one, three, and six months. Anti-SARS-CoV-2 S-RBD IgG levels were measured using the Abbott SARS-CoV-2 IgG II Quant assay (Abbott, Sligo, Ireland). We subsequently converted the AU/mL to the BAU/mL of the international standard using a conversion factor of 0.142, and analyzed the results. Section title: Study participants and evaluations Educational score: 3.9884989261627197 Domain: biomedical Document type: Study Language: en Moreover, we examined the mean CT values and areas of the deltoid muscle from CT images performed during the same period as the previous study by using image J software ( 11 ). We then compared the immunogenicity of intramuscular SARS-CoV-2 mRNA vaccination with the mean CT values or areas of the deltoid muscle. Section title: Statistical analysis Educational score: 3.7137863636016846 Domain: biomedical Document type: Study Language: en The statistical significance of the data regarding the levels of anti-SARS-CoV-2 S-RBD IgG antibodies was determined using the paired t-test. The statistical significance was set at p < 0.05 for the data analysis. Statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) ( 12 ), which is a modified version of R commander for performing statistical functions and is frequently employed in biostatistics (The R Foundation for Statistical Computing, Vienna, Austria). Section title: Characteristics of participants Educational score: 2.3506815433502197 Domain: biomedical Document type: Study Language: en Among the 75 patients in the previous study, 31 were excluded from the current study because they did not undergo CT examination during the previous study period or because the mean CT values and areas of the deltoid muscle from their CT images were not properly evaluated. We analyzed the data of 44 research participants. Section title: Characteristics of participants Educational score: 3.9028613567352295 Domain: biomedical Document type: Study Language: en Among these 44 patients, 13 had DMD (no patient treated with glucocorticoid steroids), 5 had Fukuyama congenital muscular dystrophy (FCMD), 5 had Myotonic dystrophy type 1 (DM1), 14 had cerebral palsy, 2 had spinocerebellar degeneration, 1 had sequela of brain infarction, 1 had Reye’s syndrome, 1 had Angelman syndrome, 1 had Rett syndrome, and 1 had Miller–Dieker syndrome. This study included 18 females (40.9%) and 26 males (59.1%). The median age of the 44 patients was 39 years (range: 20–63), and the mean age was 40.3 years. Section title: Anti-SARS-CoV-2 S-RBD IgG levels with respect to each factor Educational score: 3.975263833999634 Domain: biomedical Document type: Study Language: en When we analyzed anti-SARS-CoV-2 S-RBD IgG antibodies with respect to sex, age, and ambulation factors, no significant differences were observed between females (18 patients [40.9%]) and males (26 patients [59.1%]), between those aged 31 and under (17 patients [38.6%]) and those over the age of 32 (27 patients [61.4%]), and between bedridden patients (29 patients [65.9%]) and other patients (15 patients, 34.1%) ( Table 1 ). Section title: Anti-SARS-CoV-2 S-RBD IgG levels with respect to each factor Educational score: 4.06267786026001 Domain: biomedical Document type: Study Language: en Then, we divided these patients to MD (including the DMD, FCMD, and DM1) and non-MD (including SMIDs and others) groups. Twenty-three (52.3%) of the 44 patients had MDs, whereas 21 (47.7%) did not have MDs. The patients with and without MDs had median ages of 34 and 48 years, respectively, and their mean ages were 35.0 and 46.0 years, respectively. When we compared the anti-SARS-CoV-2 S-RBD IgG antibodies between patients with and without MDs, patients with MDs (mean 1,168.5 BAU/mL) showed a lower tendency than patients without MDs (mean 2,568.5 BAU/mL) at one month following the second vaccination . Although a similar tendency was also observed three months following the second vaccination ( p = 0.08), no differences between patients with and without MDs were observed during the first day of the second vaccination followed by six months post vaccination with p = 0.395 and 0.245, respectively . Section title: Anti-SARS-CoV-2 S-RBD IgG levels with respect to CT imaging Educational score: 4.0993828773498535 Domain: biomedical Document type: Study Language: en Similarly, a difference between patients with a mean CT value of zero HU and under for the deltoid muscle (mean 1,080.5 BAU/mL) and those with a mean CT value greater than zero HU (mean 2,527.0 BAU/mL) was observed at one month following the second vaccination with p -values of 0.09 . No differences were observed on the first day after the second vaccination, or three- and six-months post vaccination ( P = 0.308, 0.178, and 0.426, respectively) ( Table 1 ). In patients with and without MDs, the respective median of mean CT values of the deltoid muscle were -36.1 and 51.2 HU, and the respective mean of mean CT values of the deltoid muscle were -28.9 and 42.6 HU . Section title: Anti-SARS-CoV-2 S-RBD IgG levels with respect to CT imaging Educational score: 4.072604179382324 Domain: biomedical Document type: Study Language: en Conversely, when we compared the area of the deltoid muscle of 1000 mm 2 and under with those over the area of 1000 mm 2 , no differences were observed over time, i.e., on the first day of the second vaccination and one, three, and six months after the second vaccination . In patients with and without MDs, the respective median CT areas of the deltoid muscle were 971.2 and 1468.4 mm 2 , and the respective mean CT values of the deltoid muscle were 1014 and 1341.3 mm 2 . Section title: Discussion Educational score: 4.100371360778809 Domain: biomedical Document type: Study Language: en Our previous study showed that the immunogenicity of intramuscular SARS-CoV-2 mRNA vaccination in patients with MDs were lower than that of patients without MDs. We believed that MDs may be a significant key contributor in predicting the antibody response to intramuscular SARS-CoV-2 mRNA vaccines ( 3 ). However, the mechanism for the lower antibody response to intramuscular SARS-CoV-2 mRNA vaccination in patients with MDs remained unknown. In the current study, we indicated that the mean CT values of the deltoid muscle can account for this mechanism because a comparison of the immunogenicity of intramuscular SARS-CoV-2 mRNA vaccination between patients with and without MDs showed a similar tendency with that between mean CT values ≤ 0 HU and > 0 HU. On the basis of these findings, we posit that the quality of the muscle might affect the immunogenicity of intramuscular SARS-CoV-2 mRNA vaccination in patients with MDs. Section title: Discussion Educational score: 4.097202301025391 Domain: biomedical Document type: Study Language: en Kuru et al. ( 10 ) indicated that chronological changes in the histograms of CT values were correlated with disease progression in proportion to the degree of loss of muscle fibers and the replacement of fatty tissues; this finding is consistent with that of the current study, namely, patients with MDs have lower CT values than patients without MDs. We could not evaluate the histological effects for the antibody response to intramuscular SARS-CoV-2 mRNA vaccines because it is difficult to distinguish muscle fiber from connective tissue owing to their similar CT values ( 10 ). However, we can at least describe that the antibody response of intramuscular SARS-CoV-2 mRNA vaccines in patients with MDs may be affected by the tissue characterization of the deltoid muscle at the injection site. In addition, data on a potentially sufficient humoral immune response when administering BNT162b1 subcutaneously were also reported ( 13 ). We believe that these findings support the association of the antibody response of intramuscular mRNA vaccines and the pathological condition of patients with MDs. Section title: Discussion Educational score: 4.192147731781006 Domain: biomedical Document type: Study Language: en Three study groups reported that intramuscular SARS-CoV-2 mRNA vaccination resulted in a comfortable IgG antibody response in patients with MDs ( 14 – 16 ). Demeonbreun et al. ( 14 ) described that the presence of a normal immune response to intramuscular mRNA vaccination in patients with MDs indicated that non-muscle components significantly influence the antibody response. Iwayama et al. ( 15 ) described that intramuscular SARS-CoV-2 mRNA vaccination might increase antibody levels sufficiently via preserved vascular tissue, which allows the injected drug to reach the systemic circulation quickly even if intramuscular SARS-CoV-2 mRNA vaccines are administered to fat-replaced muscle. Saito et al. ( 16 ) reported that ambulatory status and DM1 (in addition to age) affected the low immunogenicity of intramuscular SARS-CoV-2 mRNA vaccination in patients with MDs. They mentioned that immobility and the resulting decrease in blood flow, which reduces the ability of mRNA vaccines to induce an immune response, should be considered because rich blood flow allows for the efficient processing of antigens ( 17 , 18 ). Our studies indicated that the antibody response of intramuscular SARS-CoV-2 mRNA vaccines were not affected by ambulatory status, and anti-SARS-CoV-2 S-RBD IgG levels in patients with DM1 showed low tendency. Low levels of serum IgG and abnormalities in cellular and humoral immunity in patients with DM1 have also been reported previously ( 19 , 20 ), but the mechanisms remain unclear. Taken together, we speculate that pathological condition in MDs might strongly affect the immunogenicity of intramuscular SARS-CoV-2 mRNA vaccination in patients with MDs. Section title: Discussion Educational score: 4.11722993850708 Domain: biomedical Document type: Study Language: en This study has several limitations. First, the study size was small, and no significant differences were confirmed. The immunogenicity of intramuscular SARS-CoV-2 mRNA vaccination in patients with MDs, particularly those in the MD subgroups, needs to be further investigated in a study with a larger number of cases with MDs. We speculate that disease types and pathogenesis in MDs may be important contributing factors in predicting antibody response; therefore, further studies will lead to improvements in the management of MDs. Second, the evaluation method used to assess muscles in this study was only CT imaging. We could not use several imaging techniques including MRI and dual-energy X-ray absorptiometry (DXA) ( 21 ). However, CT imaging was thought to be best because MRI and DXA have difficulty in identifying the deltoid muscle and cause contamination of artifacts. Third, as previously mentioned ( 3 ), only total IgG antibody levels against the SARS-CoV-2 S-RBD were measured in the current study. Other studies have provided not only an assessment of the humoral immune response but also combined analyses of humoral and cellular immunity. In addition, our studies have shown the association of immunogenicity with SARS-CoV-2 vaccination in patients with MDs by using only an intramuscular mRNA vaccine. Therefore, we believe that the SARS-CoV-2 immunization of patients with MDs requires extensive research. Section title: Conclusion Educational score: 3.8576061725616455 Domain: biomedical Document type: Study Language: en We reported the association of the immunogenicity of intramuscular SARS-CoV-2 mRNA vaccination with CT muscle images in patients with or without MDs. Disease type and MD pathogenesis may be key contributors in predicting the antibody response to intramuscular SARS-CoV-2 mRNA vaccine. However, SARS-CoV-2 immunization in patients with MDs still require extensive research. | Study | biomedical | en | 0.999995 |
PMC11695349 | Section title: Introduction Educational score: 3.961514711380005 Domain: biomedical Document type: Review Language: en Chrysanthemum morifolium (CM) is a globally significant ornamental plant, extensively used for culinary and medicinal purposes, with a history spanning three thousand years . Evidence suggests that CM comprises a multitude of chemical constituents, including flavonoids, phenolic acids, and lipids . This diverse chemical composition endows CM with numerous pharmacological effects, such as anti-inflammatory, antioxidant, and chronic disease prevention properties . Over time, and with its long history of cultivation, CM has been diversified into various varieties through hybridization with wild relatives and artificial breeding . In addition to its medicinal value, the ornamental characteristics of CM, particularly its wide range of colors and shapes, have contributed to its commercial and cultural significance. Section title: Introduction Educational score: 4.003998756408691 Domain: biomedical Document type: Study Language: en Significant differences exist in the composition and content of nutrients and bioactive substances among various plant varieties and their parts . CM encompasses a wide range of varieties, each exhibiting distinct variations in color, shape, and functional properties . Among these, flower color is a critical ornamental trait, significantly influencing the commercial value of CM varieties . Understanding the mechanisms that regulate flower color in CM is helpful for advancing breeding strategies and optimizing the ornamental and medical applications of these varieties. While existing studies predominantly focus on the differences in petal color among CM varieties, the non-petal parts—such as the reproductive tissues, receptacle, and calyx—have received far less attention . Although these non-petal parts may not exhibit visible differences across various CM colors, significant variations could exist at the molecular or metabolic level . Given that both petal and non-petal parts contribute to the ornamental and medicinal uses of CM, expanding research beyond petals offer helpful insights into the broader utility of this species. Thus, further investigation into the non-petal parts is warranted to uncover potential differences and their implications. Section title: Introduction Educational score: 4.090235233306885 Domain: biomedical Document type: Study Language: en The advent of high-throughput technologies, including genomics, transcriptomics, and metabolomics, has greatly advanced the exploration of the intricate molecular foundations of plant biology . These technologies have become particularly valuable in understanding the genetic and metabolic basis of traits such as flower color in CM. Metabolic profiling serves as a direct link between plant phenotypes and genotypes, identifying stage-specific metabolites and revealing the metabolic mechanisms underlying a wide variety of traits . Recent advancements in omics technologies have led to the increasing integration of metabolomics with other disciplines, such as transcriptomics. This synergy facilitates a systematic understanding of complex plant biological networks and fosters a more comprehensive biological knowledge base . Investigating the alterations in plant genes and metabolites will help elucidate the molecular mechanisms underlying the non-petal and petal parts of CM exhibiting various colors. Section title: Introduction Educational score: 4.148120880126953 Domain: biomedical Document type: Study Language: en To investigate the distinct characteristics of non-petal and petal parts in CM of varying colors and uncover potential regulatory mechanisms, white, yellow, and gold CM were subjected to transcriptomic and metabolomic analyses. Metabolomic profiling, conducted using ultra-high pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS), revealed substantial differences between the non-petal and petal components of these differently colored CM. To further elucidate the molecular basis of coloration, RNA sequencing was performed. By integrating transcriptomic and metabolomic data, the study aimed to identify key regulatory mechanisms underlying the color-dependent differences in non-petal and petal parts. This research provides new insights into the genetic basis of CM, supporting efforts to diversify CM varieties through informed breeding strategies. Section title: Materials and reagents Educational score: 3.6716651916503906 Domain: biomedical Document type: Study Language: en The CM samples gathered from the medicinal botanical garden at Hubei University of Chinese Medicine were identified and authenticated by Professor Gong Ling of Hubei University of Chinese Medicine. Fresh CM samples were immediately imaged after collection and separated into petal and non-petal tissues for subsequent analysis, as shown in Figure 1 . Non-petal samples were collected from white, yellow, and gold CM, and were designated as groups WCNP, YCNP, and GCNP, respectively. The petal samples from these colors were designated as groups WCP, YCP, and GCP. Fresh samples were stored at -80°C prior to metabolomics analysis and RNA extraction. High-performance liquid chromatography-grade acetonitrile and methanol were acquired from Merck Chemicals, Darmstadt, Germany. Section title: Sample extraction for untargeted metabolomic analysis Educational score: 4.123106956481934 Domain: biomedical Document type: Study Language: en The samples were first freeze-dried to a constant weight and then ground into a fine, uniform powder. To begin the extraction process, 100 mg of the powdered sample was weighed and extracted with 3 mL of an 80% methanol solution using ultrasonic extraction for 10 minutes. This extraction step was repeated twice: first with 3 mL of a 50% methanol solution and then with 3 mL of a 95% methanol solution. The three resulting extracts were combined, and the mixture was centrifuged at 12,000 rpm for 10 minutes. After centrifugation, the supernatant was carefully filtered and prepared for further analysis. Curcumin was employed as an internal standard in the samples at a final concentration of 200 ng mL -1 . After filtration, the quality control (QC) sample was prepared by combining equal aliquots from all individual samples. Section title: UPLC-QTOF-MS/MS-based untargeted metabolomic analysis Educational score: 4.115718841552734 Domain: biomedical Document type: Study Language: en For the analysis of the extracts, an ACQUITY UPLC H-Class system from Waters (Milford, MA, USA) was utilized, featuring a Waters ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm). The injection volume was 2.0 μL, the flow rate was maintained at 0.3 mL min -1 , and the column temperature was set to 40°C. The mobile phase consisted of two solvents: mobile phase A, a solution of water and formic acid in a 1000:1 (v/v) ratio, and mobile phase B, acetonitrile. The chromatographic gradient was programmed as follows: 0 min, 5% (B); 12 min, 35% (B); 18 min, 80% (B); 22 min, 95% (B); 25 min, 95% (B); 26 min, 5% (B); and 30 min, 95% (A) and 5% (B). Section title: UPLC-QTOF-MS/MS-based untargeted metabolomic analysis Educational score: 4.133629322052002 Domain: biomedical Document type: Study Language: en Mass spectrometry analysis was carried out using a Waters Xevo G2-XS QTOF system equipped with an electrospray ionization source. The parameters were set according to our previously published reports . The optimal parameters were as follows: desolvation temperature of 500°C, cone voltage of 20 V, capillary voltage of 3 KV, source temperature of 100°C, desolvation gas flow rate of 1000 L h -1 , collision energy range of 30 to 40 eV, and cone gas flow rate of 50 L h -1 . The mass range for full scans was set from m/z 50 to 1500 Da, with a scan duration of 1.0 s. Data acquisition was conducted in MS E mode, employing both positive and negative ion electrospray modes. Metabolomics analysis predominantly utilized positive ion mode, while structural determination of metabolites was achieved with both ion modes. Section title: RNA extraction and Illumina sequencing Educational score: 4.317534446716309 Domain: biomedical Document type: Study Language: en RNA extraction and Illumina sequencing of the samples were primarily conducted by MetWare Biotechnology Co., Ltd. (Wuhan, Hubei, China). Total RNA was extracted from plant samples using ethanol precipitation and the CTAB-PBIOZOL method, then dissolved in DEPC-treated water. RNA integrity and concentration were assessed using a Qubit fluorescence quantifier and Qsep400 biofragment analyzer (Bioptic Inc., Taiwan, China). PolyA-tailed mRNAs were enriched with Oligo (dT) magnetic beads, fragmented, and reverse-transcribed into first-strand cDNA using random hexamer primers. Strand-specific second-strand cDNA synthesis was performed with dUTPs to ensure strand specificity, followed by end repair, A-tailing, and sequencing adapter ligation. The cDNA was size-selected (250-350 bp), PCR-amplified, purified, and quantified using Qubit 4.0 (Thermo Fisher Scientific, Massachusetts, USA) and Q-PCR (Bio-rad, California, USA). Libraries were pooled based on effective concentrations and sequenced on an Illumina platform, generating 150 bp paired-end reads via sequencing-by-synthesis. Raw sequencing data were filtered with fastp, and clean reads were assembled using Trinity. Corset was then used to remove redundant isoforms from the assembled transcripts. CDS prediction was conducted with TransDecoder, and gene function annotation was performed using DIAMOND and HMMER across databases. The fragments per kilobase of transcript per million mapped reads (FPKM) for each gene were subsequently calculated based on the gene length and the number of reads mapped to it. Transcript expression levels were quantified using RSEM, with differential expression analysis conducted using DESeq2 and edgeR, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Section title: Data analysis Educational score: 4.269896507263184 Domain: biomedical Document type: Study Language: en The raw data from UPLC-QTOF-MS/MS were acquired using MassLynx v4.1 (Waters, Milford, MA, USA). Following data acquisition, the open-access software MS-DIAL (Version 4.9) was utilized for comprehensive data processing, encompassing peak detection, alignment, spectral deconvolution, identification, and normalization . The retention time for the collected peaks was set between 1 and 30 minutes. Peaks with a minimum height of 3000 and m/z values ranging from 100 to 1500 Da were selectively retained, guided by the expected component range, while the m/z value for fragment ions was set between 50 and 1500 Da. Mass tolerance parameters were established at 0.015 Da for MS and 0.02 Da for MS/MS. To determine the elemental composition of the peaks, common positive ion adducts such as [M+H] + , [M+Na] + , [M+K] + , [M+H-H 2 O] + , and [2M+H] + were employed. In negative ion mode, adducts like [M-H] - , [M+HCOOH-H] - , and [M-H 2 O-H] - were used to assist in adduct correction alongside the positive ion adducts. Feature alignment across all samples was performed with a retention time tolerance of 0.1 and an MS tolerance of 0.015. Furthermore, the deconvolution value and MS/MS abundance cutoff were set at 0.6 and 100, respectively, to facilitate peak deconvolution. The response values of the peaks were normalized using internal standards and the LOWESS method. Finally, to ensure that the screened metabolites accurately represented each group, peaks were required to be present in at least one group, with every sample within that group exhibiting the corresponding peak. Section title: Data analysis Educational score: 4.1427106857299805 Domain: biomedical Document type: Study Language: en In the search for characteristic differentially accumulated metabolites (DAMs) between two groups, we utilized variable importance in projection (VIP) values exceeding 1.25 and fold change (FC) thresholds greater than 1.5 or less than 0.67 to identify potential distinctive peaks. In the transcriptome analysis, to ensure the genes were both representative and meaningful, we applied the following criteria to eliminate false positives: (1) the average FPKM value of the gene in at least one group exceeds 1; (2) the gene’s detection rate across all samples is greater than 0.167; (3) the gene’s detection rate in any individual group is above 0.667. For the differentially expressed genes (DEGs) identified in the comparison between the two groups, the initial requirement was that genes had an FPKM > 1 in at least one group and were detected in at least two samples within any group. Subsequently, based on FPKM quantitative data, the fold change (FC) for the DEGs had to exceed 2 or fall below 0.5, with a q -value between the two groups required to be < 0.05. In the co-enrichment analysis, to identify pathways that represent both metabolomics and transcriptomics, we selected pathways containing at least two DAMs and conducted a comprehensive analysis of the DEGs upstream and downstream of the DAMs within these pathways. Section title: Data analysis Educational score: 4.055168628692627 Domain: biomedical Document type: Study Language: en The SIMCA 14.1 software (Umetrics AB, Umeå, Sweden) was employed for principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). Data visualization was carried out using Origin 2021 (OriginLab Corporation, Northampton, MA, USA) and R version 4.3.2 (R Foundation for Statistical Computing, Vienna, Austria). Statistical analyses were performed with SPSS version 26.0 (SPSS, Inc., Chicago, IL, USA). The significance of DAMs was assessed with a P -value < 0.05 using the Mann-Whitney U test, while DEGs were evaluated with a q -value < 0.05 using the Benjamini-Hochberg test. Qualitative analysis of DAMs was conducted by comparing fragment ions using literature references , MS-FINDER version 3.60 , and public databases such as the Human Metabolome Database ( https://www.hmdb.ca ) and MassBank ( https://massbank.eu/MassBank/ ). Quantitative determination of metabolites was achieved using Quanlynx software version 4.1 (Waters, Milford, MA, USA). For the co-enrichment analysis of metabolomics and transcriptomics, metabolic pathways of DAMs and DEGs were primarily constructed with reference to KEGG. Section title: Untargeted metabolomic analysis Educational score: 4.110727310180664 Domain: biomedical Document type: Study Language: en In this study, a total of 6704 features were acquired for all samples using MS-DIAL based on UPLC-QTOF-MS/MS. As shown in Figure 2A , the PCA model was applied for unsupervised analysis of all samples, including quality controls. The first two principal components accounted for 63.30% and 9.74% of the variance among the samples, respectively. The results revealed clear intra-group clustering, demonstrating strong reproducibility within each sample group. Additionally, the WCNP and YCNP groups displayed a higher degree of global similarity to each other. The clustering of the QC sample further confirmed the robust stability of instrument throughout the analysis. Section title: Untargeted metabolomic analysis Educational score: 4.138228416442871 Domain: biomedical Document type: Study Language: en Furthermore, supervised OPLS-DA was employed to identify DAMs between the two groups in both petal or non-petal areas . The scatter plots derived from OPLS-DA revealed clear segregation between each pair of groups. All pairwise comparisons yielded R2Y and Q2 scores consistently exceeding 0.9. Additionally, the results of the 999 permutation tests showed that the blue regression line of Q2 intersected the vertical axis below zero, confirming the absence of overfitting in the original OPLS-DA model. Ultimately, 90 DAMs ( P < 0.05) were characterized, with a higher number identified in the petals than in the non-petal areas. The qualitative information of these metabolites, including retention time, actual m/z mass of the quasi-molecular ion peak, tentative identification, molecular formula, secondary fragments, and classification details, was provided in Supplementary Table S1 . The FC and VIP values of DAMs across different comparison groups were listed in Supplementary Table S2 . When comparing various petal groups, WCP vs. YCP had the fewest number of DAMs, while WCP vs. GCP had the most . Meanwhile, WCNP vs. GCNP had the highest number of DAMs among the various non-petal groups. As illustrated in Figure 2C , the identified DAMs were mainly categorized into 6 categories: amino acids, flavonoid glycosides, flavonoids, lipids, other glycosides, and others. Among these, flavonoid glycosides, lipids, and flavonoids were predominantly classified in the petal parts, whereas flavonoid glycosides and lipids were primarily classified in the non-petal parts. Section title: DAMs in CMs with different colors Educational score: 4.105116844177246 Domain: biomedical Document type: Study Language: en To elucidate variations in the expression levels of these DAMs across different morphologies of CM, we conducted a relative quantitative analysis of the corresponding groups with the ratio of peak area between DAMs and internal standards, as shown in Figure 3 . In the petals of CM, the following metabolite changes were observed: 8 up-regulated and 21 down-regulated in the WCP vs. YCP, 14 up-regulated and 20 down-regulated in YCP vs. GCP, and 12 up-regulated and 25 down-regulated in WCP vs. GCP. Similarly, in the non-petals of CM, 16 up-regulated and 6 down-regulated metabolites were identified in the WCNP vs. YCNP, 5 up-regulated and 17 down-regulated in YCNP vs. GCNP, and 11 up-regulated and 16 down-regulated in WCNP vs. GCNP. Section title: DAMs in CMs with different colors Educational score: 3.9739255905151367 Domain: biomedical Document type: Study Language: en As highlighted in Figure 3 , most flavonoid glycosides showed higher concentrations in white CM, both in non-petal and petal parts. However, within the flavonoids, the petal part of white CM exhibited lower contents, while the non-petal part showed higher contents. Additionally, most lipids within DAMs were found at lower concentrations in both WCP and WCNP compared to the other two groups. Conversely, in the petals, most lipids were more abundant in YCP, whereas in the non-petal parts, they were predominantly higher in GCNP. Section title: Transcriptomic analysis Educational score: 4.130776405334473 Domain: biomedical Document type: Study Language: en To gain a more comprehensive understanding of the non-petal and petal parts across different phenotypes, transcriptomic sequencing and variance analysis of DEGs were employed to investigate the underlying molecular mechanisms. After data acquisition and transcript assembly, a total of 231670 raw sequencing reads were retained. To reduce false positives while preserving valid data, 181404 reads were selected for further transcriptomic analysis. The PCA results revealed that both yellow and gold CM showed similar trends in non-petal and petal parts compared to white CM . Moreover, each sample displayed a distinct clustering pattern within its respective group. The first two principal components accounted for 23.16% and 13.49% of the total variance among the samples, respectively. DEGs between the two groups were identified using criteria of a FC greater than 2 or less than 0.5, and a q -value less than 0.05. As illustrated in Figure 4B , the comparisons between WCP and GCP, and WCNP and GCNP, revealed the highest number of DEGs in the petal and non-petal groups, respectively. Additionally, fewer transcriptional differences were observed between yellow and gold CM. Section title: Transcriptomic analysis Educational score: 3.934475898742676 Domain: biomedical Document type: Study Language: en To further illustrate the differences between the groups, a heat map was generated from the quantitative data of DEGs, as detailed in Supplementary Figure S3 . Statistical analysis of DEG expression trends between the two groups, presented in Figure 4C , revealed that most DEGs exhibited a decreasing trend, particularly in comparisons between WCNP vs. YCNP and WCNP vs. GCNP. Section title: DEGs in CMs with different colors Educational score: 3.478459119796753 Domain: biomedical Document type: Study Language: en To systematically explore the biological functions of DEGs in petal and non-petal parts across different flower colors, we performed KEGG pathway enrichment analysis on the DEGs from each group separately. As shown in Figure 5 , the top fifteen pathways enriched by DEGs in each comparison group were selected and visualized in a bubble chart. Section title: DEGs in CMs with different colors Educational score: 4.100925445556641 Domain: biomedical Document type: Study Language: en These pathways fall into several categories, including cellular processes, environmental information processing, genetic information processing, metabolism, and organismal systems, with metabolism representing the largest portion (78.95%). Within the metabolism category, pathways related to carbohydrate and lipid metabolism were the most prevalent. Moreover, the three metabolic pathways with the highest number of DEGs were phenylpropanoid biosynthesis, pentose and glucuronate interconversions, and glycerophospholipid metabolism. Section title: Integrative analysis of CMs with different colors based on metabolomics and transcriptomics Educational score: 4.0754075050354 Domain: biomedical Document type: Study Language: en To investigate the genetic regulation of CM flower color, this study conducted a comprehensive analysis of pathways involving DEGs and DAMs, building on prior metabolomic and transcriptomic data. In order to identify pathways representing both the transcriptome and metabolome, only metabolic pathways containing at least two DAMs were retained for further analysis, with DEGs included only if located upstream or downstream of the DAMs. The KEGG codes and pathways, along with the numbers and lengths of the DEGs involved in the core pathways, were displayed in Supplementary Table S3 . Section title: Integrative analysis of CMs with different colors based on metabolomics and transcriptomics Educational score: 4.118008613586426 Domain: biomedical Document type: Study Language: en As shown in Figure 6 , glycerophospholipid metabolism and glycerolipid metabolism, both primarily involving lipids, were the primary pathways associated with CM flower color. In petal tissues, the majority of DEGs in WCP exhibited the lowest expression compared to other groups, particularly EPT1, DAD1, MGD, and psd . However, at the metabolite level, only MGDG, MGMG, and LysoPC ( sn -1) showed reduced levels in WCP. Additionally, compared to YCP, a greater number of DAMs in GCP displayed a downward trend, including LysoPC ( sn -2), PC, PE, and LysoPE ( sn -2). Section title: Integrative analysis of CMs with different colors based on metabolomics and transcriptomics Educational score: 4.056992053985596 Domain: biomedical Document type: Study Language: en Likely, in non-petal tissues, a significant number of DEGs, including MGLL, EPT1, LPCAT1_2, and DAD1, showed reduced expression in WCNP . Unlike the DAMs in petal tissues, the majority of DAMs in WCNPs exhibited significantly lower levels, particularly LysoPC ( sn -1), LysoPC ( sn -2), LysoPA ( sn -1), and LysoPE ( sn -2). Moreover, a substantial number of DAMs in GCP showed an upward trend compared to YCP, which contrasts with the pattern observed in the petal tissues. Section title: Discussion Educational score: 4.1345953941345215 Domain: biomedical Document type: Study Language: en This study investigated the impact of three distinct colors (white, gold, and yellow) on both petal and non-petal tissues of CM, utilizing an integrated approach combining transcriptomics and metabolomics to uncover the molecular mechanisms and metabolic processes associated with CM coloration. At the metabolic level, 90 DAMs were identified, predominantly flavonoids, flavonoid glycosides, and lipids. Concurrently, 38 significant metabolic pathways were enriched through transcriptomics, primarily related to metabolism, which provided potential mechanistic insights into CM coloration. By integrating metabolomic and transcriptomic data, we identified two central pathways—glycerophospholipid metabolism and glycerolipid metabolism—encompassing various lipid compounds. These pathways emerged as the primary mechanisms through which flower color influenced metabolism and gene expression in CM. Section title: Discussion Educational score: 2.037308931350708 Domain: other Document type: Study Language: en As a plant with a rich cultivation history, CM has evolved a diverse range of flower colors, including white and yellow . Flower color plays a pivotal role in shaping the aesthetic appeal and market demand for CM . Prior research on CM predominantly focused on the differential analysis of petals with notable color variations . However, our study expanded this scope, showing that varietal differences extend beyond petals to non-petal regions as well. While color variations in non-petal tissues are subtler, our results clearly demonstrated these differences. Section title: Discussion Educational score: 4.239323616027832 Domain: biomedical Document type: Study Language: en Metabolomics, which studies metabolites as the bridge between genotype and phenotype, plays a critical role in revealing plant-environment interactions . Our analysis revealed that DAMs in different CM color variants were mainly flavonoids, their derivatives, and lipids. Flavonoids and their derivatives are known functional compounds and key pharmacological constituents of CM . WCP exhibited higher levels of flavonoid glycosylation compared to YCP and GCP, with a reduction in free flavonoids and an increase in flavonoid glycosides. The decrease in flavonoids and increase in glycosylation were consistent with previous research results on white CM and yellow CM . This is likely because flavonoids contribute to vibrant coloration, offering protection against microorganisms and insects . Lipid components, essential to plant cell composition, regulate environmental adaptability . These distinctions were evident not only in petals but also in non-petal regions, suggesting that flower color may influence the physiological and functional properties of CM beyond the petals. Transcriptomics is a valuable tool in studying the genetic basis of plant diversity and addressing variations within species . Our transcriptomic analysis indicated that WCP and WCNP tissues exhibited significant differences from gold and yellow CM, with many DEGs showing a downward trend. This supported the findings of our metabolomics analysis. Section title: Discussion Educational score: 4.375913619995117 Domain: biomedical Document type: Study Language: en The integration of transcriptomics and metabolomics provided a robust approach to identifying key pathways across plant species . Our findings revealed that a significant number of DAMs and DEGs were co-enriched within the glycerophospholipid metabolism pathway, predominantly involving lipids. Glycerophospholipids, as essential components of cell membranes, played crucial roles in plant growth, development, and responses to environmental stimuli . Prior studies have noted significant differences in lipid composition among color variants, suggesting that lipid accumulation might play a key role in the coloration process . Notably, white CM showed clear differences from yellow and gold CM. Previous studies have also highlighted significant variations in the lipid composition of CM across different varieties . Our analysis further revealed that the expression levels of most DAMs and DEGs in white CM were lower than in yellow and gold CM. This suggests that the yellow and gold coloration of CM was closely associated with the synthesis of lipid metabolites and the expression of related genes. The upregulation of genes related to glycerophospholipid metabolism enhances lipid levels and increases the plant’s resilience to environmental stress . Other studies have shown that environmental changes can alter glycerophospholipid composition, modulating plant responses and potentially leading to color variation . Importantly. phospholipase A1 (DAD1) and acylglycerol lipase (MGLL) were identified as critical enzymes in mediating plant responses to both biotic and abiotic stress . Additionally, lysophospholipids accumulate in plants under stress conditions, such as freezing, injury, and pathogen infection . Since carotenoids are synthesized in plant chloroplasts and glycerophospholipid transport also occurred within these organelles, flower color might be regulated through this indirect pathway . Therefore, the environmental adaptability of CM likely regulates metabolites and genes involved in glycerophospholipid metabolism, leading to color changes. Furthermore, earlier research indicated that yellow CM exhibited superior adaptability compared to white CM, further supporting our findings . Section title: Discussion Educational score: 4.188323974609375 Domain: biomedical Document type: Study Language: en As early as the Tang Dynasty in China, only yellow varieties of CM were cultivated . Due to their high ornamental value, years of cultivation and refinement expanded their colors to include yellow, gold, white, pink, and more . Understanding the mechanisms behind this coloration had proven highly beneficial in enhancing their economic value. If the identified DAMs and DEGs were validated through further experimental verification, the expression of plant DEGs could be modulated by altering environmental conditions, introducing exogenous stimuli, or applying advanced genetic technologies based on the results. Relevant literature has reported the modification of plant color through methods such as environmental changes, introduction of exogenous interference, and gene silencing . This modulation would then regulate DAM expression, leading to the desired color. This strategy also represents a key direction for us to further explore through experiments in the future. While the most apparent differences in color lay in the petals, identifying distinctions across the entire plant, including both petals and non-petal parts, provided deeper insights into the coloration process. The integration of transcriptomics and metabolomics in this study allowed us to reveal how metabolic and genetic pathways affect non-petal tissues. These findings suggested that metabolic and genetic changes in response to flower color variation were not limited to petals, where phenotypes varied more, but may also have affected non-petal tissues. For instance, metabolic pathways involved in lipid metabolism, such as glycerophospholipid and glycerolipid biosynthesis, were found to be active in both petals and non-petal tissues, indicating that flower color regulation not only had a significant impact on the metabolic processes in petal tissues, but also on non-petal tissues. Section title: Conclusion Educational score: 4.260617256164551 Domain: biomedical Document type: Study Language: en This study employed transcriptomic and metabolomic analyses to elucidate the underlying mechanisms influencing color both in petal and non-petal tissues of CM. Pairwise comparisons of gene expression and metabolite levels across different colors revealed that lipids, flavonoids, and their derivatives were the principal metabolites affected. The glycerophospholipid metabolism, predominantly composed of lipids, and its associated gene variations emerged as crucial factors contributing to color differences in CM. Notably, significant metabolic and genetic distinctions were observed between white CM and their yellow and gold counterparts, extending beyond the petals to the non-petal tissues. Understanding the role of glycerophospholipid metabolism in flower coloration can provide a scientific basis for developing strategies to modulate flower color through environmental or genetic interventions. Furthermore, glycerophospholipids, which play a role in plant stress response and environmental adaptability, could be leveraged to breed CM varieties with improved resilience to abiotic stresses, thus contributing to sustainable cultivation practices. This research establishes a foundation for further exploration of CM coloration pathways and provides a scientific basis for quality control, cultivation, and enhancement strategies for CM. | Review | biomedical | en | 0.999995 |
PMC11695353 | Section title: 1 Introduction Educational score: 4.207496643066406 Domain: biomedical Document type: Review Language: en Diffusion Magnetic Resonance Imaging (dMRI) has established itself as a cornerstone in the study of brain microstructure, offering unmatched sensitivity for non-invasive imaging compared to conventional MRI . The advent of High Angular Resolution Diffusion Imaging (HARDI) has propelled dMRI into a new era of precision, enabling the exploration of microstructural features beyond the capabilities of traditional Diffusion Tensor Imaging (DTI) . By providing deeper insights into cellular architecture, HARDI-based approaches are particularly valuable for studying white matter (WM) development in complex scenarios, such as preterm birth, where structural abnormalities can be subtle but widespread. Section title: 1 Introduction Educational score: 3.9278671741485596 Domain: biomedical Document type: Review Language: en Despite advances in neonatal care, preterm birth remains a global challenge . Approximately 50% of survivors experience long-term neurodevelopmental impairments, including cognitive, motor, and behavioral difficulties . These outcomes are often associated with disruptions in WM integrity, which can hinder neuronal connectivity and delay brain maturation . Understanding the nature and extent of these disruptions is critical for improving diagnostics and informing targeted interventions. Section title: 1 Introduction Educational score: 4.186434745788574 Domain: biomedical Document type: Review Language: en Recent developments in dMRI have significantly improved our understanding of preterm brain development and injury, providing non-invasive insights into WM microstructure . Studies indicate that preterm birth often leads to disruptions in cortical microstructure and neuronal connectivity, contributing to developmental disabilities . While cystic periventricular WM damage has been linked to abnormal motor development, the relationship between diffuse WM damage and long-term developmental outcomes remains unclear . Advanced dMRI techniques have revealed alterations in brain region size, volume, and growth rates following preterm birth, with these changes correlated with diminished motor, cognitive, and behavioral performance from childhood into adulthood . As these imaging techniques evolve, they possess potential as biomarkers for predicting outcomes and evaluating interventions in preterm infants. Section title: 1 Introduction Educational score: 4.118776321411133 Domain: biomedical Document type: Study Language: en To unlock the broader biological implications of these findings, it is essential to integrate diverse dMRI models and innovative analytical frameworks. Based on this need, our study employs several advanced HARDI-based diffusion models to investigate preterm-related WM abnormalities comprehensively. Specifically, we focus on a variety of models that have been selected for their suitability in capturing microstructural changes beyond DTI's capabilities . These models include Diffusion Kurtosis Imaging (DKI) , Neurite Orientation Dispersion and Density Imaging (NODDI) , Multi-Shell Multi-Tissue Constrained Spherical Deconvolution (MSMT-CSD) , and FORECAST , to capture a more nuanced understanding of the WM changes associated with prematurity. Section title: 1 Introduction Educational score: 4.02996301651001 Domain: biomedical Document type: Study Language: en To explore these microstructural alterations, we adopt a dual analytical framework combining inference and prediction. Indeed, as stated in Bzdok and Ioannidis and Bzdok et al. , in the case of complex biological systems, such as the human brain, resorting to these two seemingly diverging modeling goals provides a better understanding of their complex interactions. The objective of inference entails prioritizing the contribution of each input variable through null hypothesis significance testing. In contrast, the predictive regime emphasizes on the relevance of the output of the model for accurate forecasting. Section title: 1 Introduction Educational score: 4.100842475891113 Domain: biomedical Document type: Study Language: en In this study, we employ two state-of-the-art univariate techniques representing these analytical paradigms: Tract-Based Spatial Statistics (TBSS) and Support Vector Machines (SVM). TBSS is a voxel-wise inferential method designed to detect statistically significant differences in WM microstructure across cohorts. It is widely recognized for its robustness and observer-independent nature, making it an effective tool for group-level analysis. However, its limitations in detecting diffuse abnormalities and providing personalized metrics highlight the need for complementary approaches . SVM, in contrast, represents a predictive, data-driven method that offers individualized classification capabilities. By uncovering discriminatory patterns between preterm and term cohorts, SVM bridges the gap between group-level analyses and clinical applications such as early diagnosis and prognosis . Section title: 1 Introduction Educational score: 4.098153591156006 Domain: biomedical Document type: Study Language: en Finally, to address potential redundancies in dMRI models and uncover biologically interpretable components, we also move beyond univariate analysis methods, summarizing single microstructural features at a time, toward a multivariate predictive model via Canonical Correlation Analysis (CCA) . This method integrates multiple diffusion metrics to reveal shared and distinct patterns of WM alterations . By capturing higher-order relationships among features, CCA extends beyond univariate methods such as TBSS or single-modality approaches, offering deeper insights into the complex interplay of microstructural changes in preterm birth. For further details about the three approaches, their strengths and weaknesses, and their application to this clinical scenario, please refer to Supplementary Section 1.2 . Section title: 1 Introduction Educational score: 4.137517929077148 Domain: biomedical Document type: Study Language: en To sum up, this study adopts a multi-faceted approach to understanding the biological phenomenon of prematurity through the following objectives: (i) systemic assessment through diverse dMRI models: leverage multiple HARDI-based models to explore WM microstructure comprehensively, capturing a wide range of microstructural changes beyond traditional metrics; (ii) complementary analytical strategies: combine inference (TBSS), prediction (SVM), and multimodal integration (CCA) to identify significant WM alterations, classify preterm vs. term cohorts, and uncover cross-metric relationships; (iii) bridging inference and prediction: evaluate the alignment and divergence of inferential and predictive approaches in characterizing prematurity-related WM changes, emphasizing their combined value in biomedicine. Section title: 1 Introduction Educational score: 4.059813499450684 Domain: biomedical Document type: Study Language: en By integrating these complementary analytical approaches, the present study emphasizes the importance of utilizing diverse analytical tools to uncover predictive and mechanistic insights. This all-encompassing exploration not only highlights the distinct contributions of inference and prediction but also serves as a model for tackling complex biological phenomena. This comprehensive approach positions the study as a critical step toward developing non-invasive biomarkers and personalized intervention strategies for preterm infants. Section title: 2.1 Subjects Educational score: 3.23372220993042 Domain: biomedical Document type: Study Language: en A total of 46 preterms and 23 term-born subjects were enrolled between November 2017 and August 2021 at the Neuroradiology Unit of Gaslini Children's Hospital. Conventional MRI and HARDI were performed using a 3.0 T MRI scanner (Ingenia Cx, Philips, Best, the Netherlands) with a 32-channel head array coil. Section title: 2.1 Subjects Educational score: 4.036085605621338 Domain: biomedical Document type: Study Language: en To minimize macroscopic movement artifacts, all recommended guidelines for pediatric imaging have been adopted. To protect infants from acoustic disturbances caused by MR sequences, we used baby earmuffs and silicone paste for hearing aids. Furthermore, we avoided most of the motion by swaddling infants and by placing airbags around the babies' head. In addition, protective pads have been placed between the magnet and the patient. All these factors contribute to creating a comfortable and warm rest environment, minimizing the chance of free movement. MRI was performed when possible during spontaneous sleep by administering breast milk or formula about 30 minutes before the start of the exam. In case of spontaneous sleep failure, to minimize macroscopic movement artifacts, the instrumental examination was performed under mild sedation by orally administering midazolam at 0.1-0.2 mg/kg diluted in a 33% glucose solution, subject to signature of informed consent from parents and applied by expertly trained nurses. The exclusion criteria included relevant motion artifacts, oblique positioning, an incomplete imaging process, or a low Signal-To-Noise Ratio (SNR). Section title: 2.1 Subjects Educational score: 4.086057662963867 Domain: biomedical Document type: Study Language: en Gestational Age (GA) was used as a classifying variable for preterm (GA < 37 weeks) and term birth (GA ≥ 37 weeks). We retrospectively identified all preterm neonates with birth weight <1500 g or at risk (for instance, those with anemia or intrauterine growth restriction) who underwent brain MRI at Term-Equivalent Age (TEA) in the setting of our institutional screening program for identification of prematurity-related lesions. At term, neonates underwent brain MR imaging for clinical indications, including minor trauma, suspect meningitis, and transient neurologic symptoms and signs; all had normal brain anatomy and neurologic examination. Details of the subjects demographics are reported in Table 1 . Section title: 2.1.1 Ethics approval Educational score: 1.8681448698043823 Domain: biomedical Document type: Study Language: en This single-center study was carried out in accordance with the recommendations of the Comitato Etico Regione Liguria, Genoa, Italy, with written informed parental consent obtained for each infant prior to examination in accordance with the Declaration of Helsinki. Section title: 2.2 MR acquisition Educational score: 2.724602460861206 Domain: biomedical Document type: Study Language: en Our acquisition protocol included Turbo Field Echo (TFE) 3D T1-weighted and HARDI sequences. Details of the acquisition are reported in Table 2 . Section title: 2.3.1 Structural images Educational score: 4.1408796310424805 Domain: biomedical Document type: Study Language: en The first critical step was skull-stripping. When dealing with neonatal scans, standard skull-stripping methods failed to correctly remove non-brain areas, thus requiring manual corrections and introducing both a user- and a subject-based bias. Therefore, we opted for Multi Atlas Skull Stripping (MASS) , which performs brain extraction through a template selection strategy, obtaining a higher (around 10%) accuracy than recent state-of-the-art tools and avoiding user intervention. As a preliminary step, 3D T1-weighted images were FOV-reduced, processed with Brain Extraction Toolbox (BET) , and then bias-field corrected with the N4 algorithm to suppress low-frequency inhomogeneities . At this phase, under the supervision of a board-certified neuroradiologist, we selected six subjects that best represented the anatomical variations within the dataset and processed this cohort with the developing Human Connectome Project (dHCP) pipeline . The six 3D T1-weighted brain-extracted images generated with the dHCP pipeline were subsequently used as a reference template to train the MASS algorithm. A final re-run of the N4 algorithm ensured bias-field correction using the correct mask extracted with the MASS framework instead of the rough one after preliminary brain extraction with BET. All preprocessing procedures relative to the structural scans are summarized in Figure 1A . Section title: 2.3.2 HARDI scans Educational score: 4.126349449157715 Domain: biomedical Document type: Study Language: en HARDI scans in pediatrics are sensitive to low SNR and are more prone to macro as well as micro sources of movement. We thus used Patch2Self denoising as the very first preprocessing step for diffusion imaging. This denoiser turned out to be particularly suitable for higher-order diffusion models, outperforming other existing methods at visual and modeling tasks . The method was implemented using DIPY v.1.4.0 and applied with an OLS regressor, with the threshold for b = 0 shell at 100, given the variability of non-diffusion-weighted b values. All subsequent preprocessing steps were done in Mrtrix3 v.3.0.1 . The standard analysis pipeline performed well also on neonatal scans thanks to overall good image contrast—(i) denoising; (ii) unringing; (iii) Echo Planar Imaging (EPI)—distortion correction (with reversed phase-encoding on b=0 s/mm 2 ), eddy-current and movement distortion correction; (iv) B1-field inhomogeneity correction. All preprocessing steps relative to the diffusion images are displayed in Figure 1B . Section title: 2.3.2 HARDI scans Educational score: 4.0635271072387695 Domain: biomedical Document type: Study Language: en Regarding co-registration of structural and diffusion scans, for each subject, the mean b=0 image from the diffusion data was registered to the 3D T1-weighted structural image using a rigid-body transformation in FSL , due to their high degree of overlap. The resulting inverse transformation matrix was exploited to map coordinates or data from the T1 space back to the diffusion space. This allowed subsequent analyses to be carried out in the native diffusion space of each subject, avoiding manipulation or distortion but also maintaining the inherently higher resolution of structural images. Section title: 2.3.3 Microstructural models Educational score: 4.1743083000183105 Domain: biomedical Document type: Study Language: en All quantitative diffusion features in this study result from fitting a different model to the measured dMRI signal on a voxel-wise basis. Despite the multiplicity of existing microstructural dMRI models, the majority fall under the category of linear models fitted with linear least-squares, hence the redundancy of information concealed in diffusion measures. More specifically, all these models share the representation of the dMRI signal as an expansion in an appropriately chosen functional basis, where the coefficients are determined using some variation of least squares . In virtue of this, in the absence of noise, they all can be traced back to the same mathematical equation: Section title: 2.3.3 Microstructural models Educational score: 3.8915536403656006 Domain: biomedical Document type: Study Language: en where y is the response variable to be modeled, x is a single measurement, and ϕ i ( x ) is the (possibly nonlinear) function with the corresponding coefficients c i . In practice, given N observations ( x j , y j ), we aim to estimate c ^ such that y ≈ Φ c ^ , where we have introduced the matrix Φ ji = ϕ i ( x j ). Section title: 2.3.3 Microstructural models Educational score: 2.4873766899108887 Domain: biomedical Document type: Study Language: en Multivariate CCA analysis has been applied precisely to further investigate how each of these diffusion features relates to each other, given their common starting mathematical formulation . Section title: 2.3.3 Microstructural models Educational score: 4.057465553283691 Domain: biomedical Document type: Study Language: en These microstructural dMRI models have been easily utilized for this cohort thanks to the overall high image quality . The outcome produced by each model has been inspected by two experienced pediatric neuroradiologists (DT and MS) with 10 and 15 years of experience, respectively, and compared with existing studies on age-matched cohorts. Furthermore, to avoid spurious contributions from non-representative image portions and to reduce computational time, all models have been applied to a masked version of the data derived from averaging and skull-stripping the non-diffusion weighted pre-processed volumes. Further details about each specific HARDI microstructural model are provided in Supplementary Section 1.1 . Section title: 2.3.3.1 Diffusion Kurtosis Imaging Educational score: 4.0226616859436035 Domain: biomedical Document type: Study Language: en We estimated DKI maps using DIPY v.1.4.0 ( https://dipy.org ) . Standard parametric maps—Mean Kurtosis (MK), Axial Kurtosis (AK), Radial Kurtosis (RK), and Kurtosis Fractional Anisotropy (KFA)—were thus generated. Since these measures are susceptible to high-amplitude outliers, we removed their impact by limiting the extraction of metrics within the typical range (0, 3). Section title: 2.3.3.2 Neurite orientation dispersion and density imaging Educational score: 4.097480773925781 Domain: biomedical Document type: Study Language: en We computed NODDI-related measures—Intra Cellular Volume Fraction (ICVF), ISOtropic Volume Fraction (ISOVF), and Orientation Dispersion Index (ODI)—with a linear framework for Accelerated Microstructure Imaging via Convex Optimization (AMICO) implemented in Python ( https://github.com/daducci/AMICO ), which, through a convex optimization approach, drastically accelerates the fit of advanced dMRI techniques while preserving accuracy and precision in the estimated parameters, thus meeting real application demands . Section title: 2.3.3.3 Fiber Orientation Estimated using Continuous Axially Symmetric Tensors Educational score: 4.136556625366211 Domain: biomedical Document type: Study Language: en We resorted to DIPY also for the computation of measures derived from the FORECAST model . We used 6 as the spherical harmonics order ( sh _ order ) for the fiber Orientation Distribution Function (fODF) and CSD as the spherical deconvolution algorithm for the FORECAST basis fitting ( dec _ alg ) to extract crossing invariant tensor indices. These are mean diffusivity (md), perpendicular diffusivity (d ⊥ ), parallel diffusivity (d ∥ ), and fractional anisotropy (FORECAST-fa). Using all b-value shells with a basis order of 6 fully leverages the available diffusion-weighted information across varying diffusion sensitivities. This configuration effectively captures both large-scale orientations and fine microstructural details, making it well-suited for robust and computationally efficient studies of complex white matter architecture . Section title: 2.3.3.4 Multi-Shell Multi-Tissue Constrained Spherical Deconvolution Educational score: 4.157501220703125 Domain: biomedical Document type: Study Language: en Application of MSMT CSD has been performed in MRtrix3 ( http://www.mrtrix.org/ ). For response function estimation, used as the kernel by the deconvolution algorithm, we resorted to the dhollander approach, suitable for computing MSMT response functions in the case of multi-tissue variants of SD and more reliable in the case of neonates . We also maintained the default spherical harmonics order in MRtrix3's MSMT CSD implementation to achieve an optimal balance of angular resolution and noise resilience. This choice aligns with best practices for neonatal HARDI data and leverages MRtrix3's robust, validated parameter defaults to ensure consistency and reliability in diffusion modeling . However, given the poor WM/Gray Matter (GM) contrast inherent to neonatal scans , we were limited to extracting tissue-specific ODF just for WM and Cerebro-Spinal Fluid (CSF). Moreover, since we were interested in performing population studies, we used the same response function for all our cohorts. To this end, we calculated the average tissue response function for all subjects exclusively for WM and CSF responses, named wm and csf, respectively. Section title: 2.3.4.1 FA skeleton generation Educational score: 4.082212924957275 Domain: biomedical Document type: Study Language: en We first used TBSS, a widely used voxel-wise statistical inference for WM anatomy , to inspect potential per-voxel differences across microstructural-derived markers typical of preterm birth compared to term-born controls. However, once again neonatal imaging caused the standard TBSS pipeline developed in FSL ( https://fsl.fmrib.ox.ac.uk/fsl/fslwiki ) to present technical challenges due to smaller anatomical dimension and lower image contrast and resolution. We thus integrated it with DTI-TK ( http://dti-tk.sourceforge.net/pmwiki/pmwiki.php?n=Documentation.TBSS ), as suggested also in Bach et al. and Tokariev et al. . Section title: 2.3.4.1 FA skeleton generation Educational score: 3.286747694015503 Domain: biomedical Document type: Other Language: en The latter is a spatial normalization and atlas construction toolkit optimized for examining WM morphometry through tensor-based registration able to leverage rich discriminating features. Section title: 2.3.4.1 FA skeleton generation Educational score: 4.077327728271484 Domain: biomedical Document type: Study Language: en The main differences from the standard TBSS pipeline are: (i) limiting DTI tensor computation through FSL to the b =700 s/mm 2 shell rather than the whole multi-shell diffusion volume; (ii) at the registration phase, bootstrapping a population-specific DTI template from our whole cohort of study without relying on an existing one to better capture within-population features ; (iii) thresholding the resulting WM skeleton of the high-resolution population-specific DTI template at 0.1 level, in agreement with other works on neonates . Section title: 2.3.4.2 Non-FA metrics Educational score: 4.113237380981445 Domain: biomedical Document type: Study Language: en In order to extend TBSS analysis to diffusion-derived measures other than DTI-FA, we repeated DTI-TK + TBSS steps, similar to what was done in Timmers et al. . Specifically, these non-FA metrics include DKI- (MK, AK, RK, KFA), NODDI- (ICVF, ISOVF, ODI), FORECAST- (md, d ∥ , d ⊥ , FORECAST-fa), and MSMT CSD (wm, csf)—derived measures, respectively. We thus converted each microstructural scalar map to the DTI-TK coordinates, and then we reapplied to each measure the previously obtained nonlinear registration transform to transfer each DTI-FA map to the population-specific template. This procedure was repeated for each of the microstructural measures analyzed in this study. Section title: 2.3.5.1 Machine Learning methods for classification Educational score: 2.3156168460845947 Domain: biomedical Document type: Study Language: en Moving to ML analysis, we performed preterm/term-born subject classification based on a predictive model. Section title: 2.3.5.1 Machine Learning methods for classification Educational score: 3.993722677230835 Domain: biomedical Document type: Study Language: en Given the small amount of data available to train our model, we thus resorted to an SVM framework to categorize preterm-born and term-born individuals based on the whole-brain WM skeleton estimated using TBSS. Indeed, among the variety of predictive techniques applied so far in neuroimaging settings, SVM has emerged as one of the most effective methods in coping with high-dimensional data and providing good classification results . Section title: 2.3.5.1 Machine Learning methods for classification Educational score: 2.216240644454956 Domain: biomedical Document type: Study Language: en We also carried out a further analysis to investigate how the performance changes by varying the input dimension of our data through feature selection, and then we trained a classification model based on related findings. For the implementation of ML methods, we resorted to scikit - learn free software in Python ( https://scikit-learn.org/stable/ ). Section title: 2.3.5.2 Experimental design Educational score: 1.5238035917282104 Domain: biomedical Document type: Study Language: en The experiments we carried out can be subdivided into two phases . Section title: 2.3.5.2 Experimental design Educational score: 4.211885452270508 Domain: biomedical Document type: Study Language: en In the first phase, we adopted SVM to perform binary classification starting with the DTI-FA map, computed through DIPY v.1.4.0, warped to common TBSS space, and masked by the thresholded WM skeleton for all 69 infants involved. We then split the dataset into learning and testing by stratified 5-fold cross-validation ( outer - CV ) to increase the numerosity of our dataset while preserving the same class ratio throughout the K folds as the ratio in the original dataset. For each fold, we thus applied data normalization in the default range on both the learning set and the test sets. We then further split the learning set into training and validation sets, named inner - CV , to exhaustively tune the model hyperparameters with the GridSearchCV instance. We thus looked for the best hyperparameter grid by choosing the one that produced the lowest prediction error. This set included: (i) the best penalty term C (among 0.001, 0.01, 0.1, 1, 10, 100, and 10 6 ); (ii) the best kernel (among linear, radial basis function and polynomial with default degree=3); and (iii) the optimal number of features (selecting 20%, 40%, 60%, 80%, and 100% of the input dataset with the SelectKBest method). For each combination of hyperparameters, we fitted a model on the training set and thus evaluated its performance by computing the average F1 score across folds on the validation set. By selecting the set of parameters whose average F1 score was the best, we then trained such an SVM model on the learning set and subsequently evaluated its performance in terms of average and standard deviation of accuracy, precision, recall, F1 score, and Area Under the Receiver Operating Characteristic (ROC) curve (AUC) across folds on the unseen test set. Section title: 2.3.5.2 Experimental design Educational score: 4.087884426116943 Domain: biomedical Document type: Study Language: en In the second phase, once selected the model classifier offering the best performance on DTI-FA data was identified, we further evaluated the classification performance when giving as inputs the parametric measures from other microstructural models than DTI. In this phase, we did not perform any inner - CV as we did not introduce a hyperparameter search. The decision not to re-optimize the classifier was justified by the desire to maintain a controlled comparison between the series of measurements, using the hyperparameters from the first stage. Indeed, this approach minimizes variability by focusing on how different microstructural measures affect model performance. Conversely, for each input variable, we again carried out the outer - CV to provide a more robust evaluation of the model. We thus trained the model on the learning set and then assessed the model on the test set, computing the average and standard deviation of usual scores. Section title: 2.3.6 Weight maps extraction and comparison with TBSS Educational score: 4.083222389221191 Domain: biomedical Document type: Study Language: en Finally, to relate the results from inferential speculation with those from prediction, we extracted weight maps from the selected SVM classifier within outer - CV , averaged them across the 5 folds, normalized them between 0 and 1, and reshaped them as the 3D input TBSS skeleton for mere visual comparison. The weights are SVM coefficients determining the discriminant hyperplane, which depicts the relevance of each voxel for classification between positive and negative conditions. Section title: 2.3.6 Weight maps extraction and comparison with TBSS Educational score: 4.06840181350708 Domain: biomedical Document type: Study Language: en We thus computed the standard Pearson's correlation between the normalized SVM weight maps and TBSS normalized significance maps ( p -maps) for each of the microstructural measures analyzed. To further inspect the overlap between WM discriminating features detected by ML and TBSS, we related Pearson's correlation with the Wasserstein Distance (WD) metric to quantify the distance between the two distributions. Both measures have been computed via the Python library scipy . Section title: 2.3.7 Multivariate predictive model Educational score: 3.988431215286255 Domain: biomedical Document type: Study Language: en Moving to multivariate analysis, the CCA method is based on establishing linear relationships between two or more sets of variables to find out inter-subject co-variances. CCA looks for two or more sets of transformed variates—Canonical Components (CCs) or Variates (CVs)—to assume maximum correlation across the two datasets while being uncorrelated within each dataset. Details about its mathematical formulation are provided in the Supplementary Section 1.2 . Section title: 2.3.7 Multivariate predictive model Educational score: 4.076096534729004 Domain: biomedical Document type: Study Language: en In our study, we resorted to the open-source Python package Pyrcca to perform a multi-set CCA based on fusing all advanced dMRI models under analysis . We used as input all 14 HARDI measures after filling in missing values and z-scoring. A linear kernel was used to reduce the computational complexity of the analysis. Moreover, we opted for a regularized kernel CCA to avoid overfitting, given the low numerosity of our datasets, and to relax the orthogonality constraint between the CCs. Finally, we estimated the optimal set of CCA hyperparameters—the regularization coefficient and the number of CCs—empirically by using GridSearchCV . Specifically, the optimal regularization parameter was chosen from a logarithmically spaced range of 10 values between 1 × 10 −4 and 1 × 10 2 , while the optimal number of components was chosen between 1 and 5. We selected these ranges based on pilot analyses performed on an independent dataset that was not used for this publication. Section title: 2.3.7.1 Shared/distinct abnormalities Educational score: 4.061975002288818 Domain: biomedical Document type: Study Language: en As in Sui et al. , we inspected group differences between the two cohorts by performing a non-parametric Mann-Whitney U Test between each pair of CCs to look for the variates showing abnormalities associated with preterm birth. The statistical survey was followed by the Benjamini-Hochberg correction method for multiple comparisons . If the components show group differences in more than one dMRI model, they are called modality-common or joint group-discriminative CVs. Conversely, if the components show group differences only in a single model, they are called modality-unique group-discriminative CVs. Section title: 2.3.7.2 Inter-modality correlation Educational score: 4.058840751647949 Domain: biomedical Document type: Study Language: en We then investigated the inter-correlation existing between microstructural dMRI models by looking at the Canonical Correlation Coefficients (CCC) to establish whether the joint-group discriminative components additionally have strong inter-modality correlation, which would reflect the interaction and correspondence among diffusion imaging techniques. Section title: 3.1 TBSS analysis exhibits a significant decrease in preterm subjects for a subgroup of HARDI measures Educational score: 4.1871771812438965 Domain: biomedical Document type: Study Language: en Cross-subject voxel-wise TBSS statistics unraveled significantly different voxels exclusively on a subset of the microstructural maps under consideration, using an unpaired voxel-wise t-test with Family-Wise Error (FWE) correction using Threshold-Free Cluster Enhancement (TCFE) . Specifically, compared with the term cohort, the preterm group showed a significant decrease in DTI-FA, MK, AK, ICVF, and FORECAST-fa. The WM regions with significant between-group differences in diffusion metrics are shown in Figure 6 . Conversely, no significant differences were observed by TBSS analysis in RK, KFA, ISOVF, OD, MD, d ∥ , or, d ⊥ , or in MSMT-derived measures. Table 3 summarizes the significant clusters identified by TFCE in WM regions where diffusion metrics showed decreased values in the preterm group compared with the term group, highlighting metric-specific spatial patterns and degrees of sensitivity to WM microstructural alterations associated with prematurity. Section title: 3.1 TBSS analysis exhibits a significant decrease in preterm subjects for a subgroup of HARDI measures Educational score: 4.169158935546875 Domain: biomedical Document type: Study Language: en More in detail, compared with the term group, the preterm cohort had significantly decreased DTI-FA values in widespread WM areas, predominately in the genu, body, and splenium of the corpus callosum; right internal and external capsule, corona radiata, and posterior thalamic radiation. The distribution of areas with decreased MK was similar with respect to the areas with decreased DTI-FA. AK exhibited a pattern analogous to MK whilst comprising a bilateral external capsule. The same applies to the ICVF metric. The amount of WM areas showing a significant decrease in prematurity increased for the FORECAST-fa parameter, which extended to the whole corpus callosum, bilateral internal capsule, external capsule, anterior corona radiata, and, finally, posterior thalamic radiation (including the optic radiation). Section title: 3.2 SVM classification of group membership achieves good performance, especially in terms of area under the curve score Educational score: 1.9004591703414917 Domain: biomedical Document type: Study Language: en Since the performance of a model significantly depends on the value of its hyperparameters, we first focused on hyperparameter tuning to determine the optimal values for our classification estimator. Section title: 3.2 SVM classification of group membership achieves good performance, especially in terms of area under the curve score Educational score: 4.030421733856201 Domain: biomedical Document type: Study Language: en In this respect, Figure 7A shows the result of the cross-validated grid search over the parameter grid across each of the five folds. Furthermore, based on the selected hyperparameters, we fitted our model on the training set and evaluated its performance on the test set in terms of F1 score, accuracy, precision, recall, and AUC across each of the five-folds . To establish the best estimator possible based on the input data, we counted how many folds in which a variable was selected and could thus conclude that penalty term C and linear kernel were the most frequently selected hyperparameters. Conversely, the search turned out to be less stable in terms of the optimal number of features, which varied at every fold . Therefore, to set the last missing parameter for our estimator, we set C and kernel according to their most chosen values while varying the number of features as a percentage of the total amount. Section title: 3.2 SVM classification of group membership achieves good performance, especially in terms of area under the curve score Educational score: 4.158938407897949 Domain: biomedical Document type: Study Language: en Figure 7C confirms that, in our case, feature selection is not beneficial for improving classification performance. Indeed, both average value and standard deviation across folds of each score remain constant with variable subsets of features. In addition, the average AUC score proves to be maximal (0.87) when including the whole feature amount. We thus opted for avoiding feature reduction and kept the whole of the features to define the final version of our SVM estimator. As regards this definitive version of the classifier, a detailed plot of the ROC curve profile for every fold is displayed in Figure 7D . We subsequently trained a classification model without hyperparameter search ( inner - CV ) using as input variables the metrics derived from other microstructural HARDI models. Performance in terms of F1 score, accuracy, precision, recall, and AUC for the whole set of microstructural parameters, including DTI-FA, is reported in Figure 8 . Of note, among the whole set of measures, the ones exhibiting the highest discriminative power in terms of SVM classification are those probing overall anisotropy and directionality of fibers, namely DTI-FA, KFA, OD, and FORECAST-fa, for which all scores overcome 75%, 74%, 70%, and 74% levels on average, respectively. Section title: 3.3 Comparison between SVM and TBSS reveals a measure-dependent rate of agreement between the two approaches Educational score: 4.171626091003418 Domain: biomedical Document type: Study Language: en Relating variables identified as statistically significant with those identified as predictively relevant, a statistically significant Pearson's correlation for all microstructural measures considered ( p < 10 −2 ) (see Table 4 ) arose. This relationship was further confirmed by inspecting the association between the absolute Pearson's correlation coefficient and WD, reported in Figure 9 , showing a trend of indirect proportionality. Overall, an inverse trend was observed, with measures showing relatively higher absolute correlations generally corresponding to lower WD, although this relationship may vary slightly across specific measures. The correlation was moderate ( r = 0.61) for the d ∥ parameter, weakly moderate ( r ∈ 0.45 − 0.51) for RK, KFA, DTI-FA, and OD, low ( r ∈ 0.28 − 0.35) for MK, AK, MD, FORECAST-fa, and ICVF, and very low ( r ∈ 0.05 − 0.14) for d ⊥ , CSD-related measures, and ISOVF . These results suggest an overall good, though measure-dependent, rate of agreement between p -maps derived by the TBSS approach and weights probing the discriminative power of SVM. Section title: 3.4 Canonical Correlation Analysis unravels joint group differences for parallel diffusivity and ISOtropic Volume Fraction Educational score: 4.161378860473633 Domain: biomedical Document type: Study Language: en Moving to CCA analysis, Pyrcca cross-validated hyperparameters search detected the optimal regularization coefficient equal to 0.01, and the optimal number of CVs to 4. Preliminarily, the results of CCA analysis were evaluated in terms of Canonical Correlations to determine the number of meaningful CCs recovered by Pyrcca. Figure 10 contains a heatmap of pairwise correlations between the 14 HARDI measures for each of the 4 sets of CCs. From the Mann–Whitney U Test, CCA analysis applied to our cohort unraveled group differences in the 4th Canonical Component, for which statistically significant differences between preterm and term subjects have been found in ISOVF and d ⊥ even after outlier removal with the interquartile range method and FDR correction ( p = 0.014, U = 621 and p = 0.014, U = 759, respectively, α = 0.05), thus being a joint group discriminative independent component. This is depicted in Figure 11A , with violin plots of CVs having statistically significant differences between preterm and term-born subjects. Interestingly, the intramodal connection within the joint-discriminative independent component (4th) indicates a good correlation ( r = 0.62) . Furthermore, to visually mark out detected differences between the two groups, we displayed derived spatial maps only for the specific joint group-differentiative CC. In Figure 11B , each z-score-transformed input measure is reported to highlight statistically significant group-discriminating subsets of voxels. Section title: 4 Discussion Educational score: 4.1427507400512695 Domain: biomedical Document type: Study Language: en In this work, we examined the complexities associated with preterm birth through a multiplicity of advanced HARDI models in turn employed at different levels of analysis. To the best of our knowledge, this study is thus the first to jointly employ univariate statistics (TBSS) and predictive modeling (SVM) on intramodal advanced dMRI data to comprehensively investigate WM alterations associated with preterm birth. Unlike prior works that have predominantly used these methods independently, our approach leverages their complementary strengths—TBSS for robust group-level analysis and SVM for individualized prediction—providing a multifaceted perspective on WM microstructure. Additionally, for the first time, we integrate CCA to uncover hidden relationships among multiple advanced dMRI models, surpassing traditional statistical tools in capturing complex interdependencies. By combining these methodologies, our study represents a significant advancement toward identifying biologically interpretable and clinically relevant markers of WM alterations in preterm infants, paving the way for more personalized and data-driven approaches in neonatal neuroimaging. Section title: 4 Discussion Educational score: 4.286027431488037 Domain: biomedical Document type: Study Language: en The first tool we considered to investigate the potential characteristics of preterm subjects was TBSS. Through this inferential data analysis strategy, we demonstrated that both DTI-FA and non-FA values can be useful measures to distinguish relevant WM tracts in preterm-born neonates at TEA from term-born controls. It was particularly notable that there was a correspondence between the distribution of areas with decreased DTI-FA and non-FA measures, with an expansion of WM-discriminating areas over the main tracts, especially in the case of beyond-DTI measures. This agrees with existing findings in the literature claiming that: (i) WM maturation is associated with increasing axonal organization, pre-myelination, and myelination, which progressively restricts water diffusion perpendicular to the direction of the axonal fiber; (ii) since premature birth may lead to relatively slow brain development in premature infants, some brain regions are less developed than the full-term infants. This includes the corpus callosum, anterior and posterior limb of the internal capsule, and, more generally, all tracts subject to early myelination whose metabolism is thus vigorous and the oxygen demand is high, which makes these metabolically active areas the first to be damaged in case of risk factors for preterm birth . For the DTI measures, lower FA has been found across the WM in preterm infants compared with term-born infants , which correlated with increased prematurity . Furthermore, WM diffusion measures in preterm infants at TEA have been related to subsequent neurodevelopmental performance. Decreased DTI-FA measures, with an expansion of together with increased MD and RD-FA measures, with an expansion of in the WM at TEA are associated with worsened motor, cognitive, and language performance in early childhood as well as visual function . In Zhao et al. , kurtosis-related parameters, especially MK, were shown to sensitively reflect the brain maturity of premature infants. Decreased MK values were registered in the preterm cohort due to the decreased density of cells and axon membranes associated with impaired brain development. Section title: 4 Discussion Educational score: 4.162930488586426 Domain: biomedical Document type: Study Language: en Similarly, the NODDI model has been applied to investigate WM and GM maturation in the preterm brain , finding that ICVF increases in the WM with increasing maturation, mainly attributed to increasing axonal growth/density/packing/diameter or pre-myelination/myelination changes, rather than changes in axon coherence or geometry. Moreover, greater ICVF in childhood has been associated with better neurodevelopmental outcomes, IQ , visual motor integration , motor, behavioral, and emotional scores , language , and maths . Finally, although not previously investigated in the case of preterm subjects, the FORECAST-fa parameter falls into those measures exhibiting significant differences from preterm to term-born infants, presumably for being the equivalent of the DTI-FA yet far more sensitive to the underlying fiber microanatomy. Section title: 4 Discussion Educational score: 4.245734214782715 Domain: biomedical Document type: Study Language: en The second perspective from which we examined our cohort was an SVM-based approach aimed at a more individualized classification method to overcome shortcomings of group-wise investigations. The good achievement of the SVM in correctly assigning group membership, based on a single MR image, indicates that the distinct brain development of preterm-born individuals can be successfully identified by predictive methods. Indeed, considering the low sample size at disposal, much inferior to the number of features (i.e., image voxels), the SVM classifier managed to handle the issue of overfitting and proved a good performance on both the DTI-FA skeletonized image, on which its model was designed, and on the vast majority of non-FA measures. Specifically, together with DTI-FA, other scalar parameters derived from DKI, NODDI, and FORECAST exhibited good scores in terms of both F1/accuracy and, most importantly, of AUC—a significantly more meaningful measure of classifier performance than accuracy because it does not bias on size of test or evaluation data. Of note, preterm vs. term classification accuracy achieved by the predictive model, however good, was not optimal. This may be due to the diffuse effect of preterm birth on WM microstructure, being optimally captured by methods not requiring anatomically constrained ROIs . Along with overall good performance scores, the selected classifier also showed strong robustness (i.e., limited variability across folds), another important indicator for model evaluation, assessing its stability. Furthermore, the evidence that the most discriminating features in terms of SVM classification are related to fiber anisotropy stands for dysmaturation or delay in myelination of WM tracts following preterm birth in contrast to term-born controls. Section title: 4 Discussion Educational score: 4.0933308601379395 Domain: biomedical Document type: Study Language: en We then explored the relationship occurring between TBSS- and SVM-based methods, assessing the degree of overlap between the two survey methods in attributing relevance to the input variables. The observed negative Pearson's correlation is explained by considering that we compared a significance map from voxel-wise statistics made up of thresholded p -values and the map of SVM weight vectors serving as a ranking metric for measuring feature importance . As a result, voxels exhibiting a lower p -value correspondingly have a high ranking in the SVM model, which results in the observed inverse trend. Section title: 4 Discussion Educational score: 3.9539973735809326 Domain: biomedical Document type: Study Language: en The partial agreement between voxel features identified by TBSS and SVM reflects the complementary strengths of inferential and predictive methods. TBSS excels at identifying group-level differences, while SVM highlights individualized discriminative features, providing a multifaceted understanding of WM alterations. Section title: 4 Discussion Educational score: 4.097862243652344 Domain: biomedical Document type: Study Language: en Such findings are in line with Bzdok et al. , who directly compared explanatory and predictive modeling in both simulated and common real-world datasets, finding out a certain variability in feature identification between the two approaches, with increasing divergence in typical clinical settings. This discrepancy is attributable to the specific data scenario at hand, including properties of the data-generating mechanisms (e.g., available sample size, number of informative input variables, redundancy of information carried in the input variable about the outcome, random noise variation, pathological settings), which affect variable identification in TBSS and SVM in distinct ways. Specifically, small-to-moderate sample size and collinearity between input measures, very common in biological data such as in our case, together with the number of truly relevant variables, commonly unknown in biomedical data analysis in practice, are those driving experimental factors causing the largest disagreements in variable identification. Section title: 4 Discussion Educational score: 4.227879047393799 Domain: biomedical Document type: Study Language: en It is on the premise that integrating multiple datasets from the same participants can increase confidence when making conclusions to a greater degree than traditional statistical approaches that we extended our investigation to considering simultaneously multiple microstructural models through CCA. In this study, we investigated brain co-alterations from several advanced dMRI across preterm and term-born cohorts. To our knowledge, this is the first study to clarify preterm birth-related brain changes in different dMRI modalities via an intramodal data fusion model. Specifically, two further measures, d ⊥ and ISOVF, emerged as relevant markers discriminative of preterm birth, other than those highlighted by TBSS or classification. This proves the capability of CCA to detect potentially hidden relationships between different imaging modalities beyond traditional methods, which could not be detected from a single dMRI model. The brain regions exhibiting the strongest contributions to coherent changes related to preterm birth involve the majority of WM tracts detected with TBSS. More in detail, a simultaneous decrease in both d ⊥ and ISOVF is observed in the term-born group compared to the preterm one. Such findings are in line with previous studies and consistent with the higher content of extracellular free water expected in the case of diffuse loss of WM microstructural integrity and organization inherent to preterm birth. Similarly, being d ⊥ a more sensitive variant of DTI's RD, it proved in turn to be highly reflective of the lack of tortuosity imposed on water motion due to the delayed development of the myelin sheath . Section title: 4 Discussion Educational score: 4.109744548797607 Domain: biomedical Document type: Study Language: en Through this investigation, we highlight how “importance”, intended as variable relevance, does not have an unambiguous definition across different data analysis strategies. Resorting to p values or prediction accuracies for arguing research claims both have flaws and each is insufficient per se. Our findings thus push toward the adoption of a combined approach aimed at exploring similarities and differences of significance and predictability to fully exploit the advantages of both methods in the perspective of a patient-tailored predictive approach, where the goal of forecasting patient-specific disease symptoms in turn helps and complements explaining disease-causing biological mechanisms, finding a common ground between these two apparently opposed methods. In this sense, inference and prediction can be seen as two sides of the same coin, both aimed at understanding and using data to make informed and better decisions. Section title: 4 Discussion Educational score: 3.8926608562469482 Domain: biomedical Document type: Study Language: en We are aware that both voxel-wise statistical methods and, in particular, the ML approach benefit from large quantities of data. Our survey is inherently limited by the challenge of collecting clinical data from the targeted population and thus provides preliminary, exploratory insights into microstructural changes associated with prematurity. However, all methodological strategies to handle this issue have been adopted to balance interpretability with predictive robustness, such as the choice of SVM as a classifier and the implementation of nested cross-validation. Section title: 4 Discussion Educational score: 3.826662063598633 Domain: biomedical Document type: Study Language: en Future studies will focus on extending the current dataset and introducing stratification by diagnosis, which could enhance the predictive classification's ability to detect specific WM tract patterns across healthy and pathological preterm subjects. This aligns with our broader goal of developing clinically actionable tools for understanding and monitoring prematurity-related WM changes. Section title: 5 Conclusion Educational score: 4.155602931976318 Domain: biomedical Document type: Study Language: en Results gathered so far from this study revealed that an intramodal dMRI approach can be a valuable tool to distinguish atypical brain microstructure at TEA when compared with a full-term group, regardless of the specific diagnosis based on radiological findings. This differentiation is achieved at three different levels of investigation, to provide a more comprehensive, detailed, and biologically meaningful interpretation of WM microstructure changes associated with prematurity. First, a classical group-level survey tool such as TBSS confirmed the high sensitivity of advanced dMRI methods. Second, a state-of-the-art approach based on SVM classification achieved a high recognition rate. Furthermore, comparing the two methods revealed a distinct agreement in selecting the most discriminating WM regions, mainly depending on the microstructural measure under consideration. Finally, CCA further represents a powerful tool for identifying the inter-measure similarities between metrics associated with preterm birth in a data-driven way, without imposing an explicit model. Section title: 5 Conclusion Educational score: 3.8772432804107666 Domain: biomedical Document type: Study Language: en Taken together, these insights suggest that combining synergy between modalities and analytical tools will allow for a more thorough investigation of the preterm birth phenomenon providing an unprecedented supplement to our understanding of biological mechanisms. Furthermore, these findings should be added to the body of literature suggesting that there is generalized dysmaturation of the WM in preterm neonates. Section title: 5 Conclusion Educational score: 2.5343313217163086 Domain: biomedical Document type: Other Language: en Further studies should focus on investigating how well these results generalize to data across centers and on what kind of improvements are needed to reach the end goal of predicting, on an individual basis, the specific outcome of subjects born preterm. | Review | biomedical | en | 0.999997 |
PMC11695354 | Section title: 1 Introduction Educational score: 4.02711820602417 Domain: biomedical Document type: Other Language: en Spiking Neural Networks (SNNs) are the third generation of artificial neural networks , inspired by the functioning of biological neurons. Unlike traditional neural networks, which are stateless and process information through continuous activation values, neurons in SNNs are stateful and communicate via sparse binary spikes, mimicking the electrical impulses observed in biological neurons. This enables SNNs to efficiently process temporal information, making them well-suited to tasks involving sequential data processing. SNNs have shown significant potential as a computational paradigm for neuromorphic computing platforms , enabling low-power and real-time processing. This makes them a compelling basis for next-generation intelligent systems. Section title: 1 Introduction Educational score: 4.261472702026367 Domain: biomedical Document type: Study Language: en Synaptic delays (between the emission of a spike and its arrival at the post-synaptic neuron) have been suggested as a means of improving the spatio-temporal information processing of SNNs . These delays, which can vary across connections, allow neurons to perform coincidence detections across longer time intervals, enhancing SNN's ability to process temporal information. In biological systems, delays encompass axonal, synaptic, and dendritic components and are modified by processes like myelination to facilitate learning and coincidence detection . Furthermore, neuromorphic hardware such as Intel's Loihi , SpiNNaker , and DenRAM incorporate programmable synaptic delays, enabling SNNs with delays to be efficiently deployed for real-time data processing. In the past, two kinds of delay learning methods have been used: delay selection and delay shift. Delay selection relies on implementing several synapses between each neuron with various delays, and picking the most optimal one . Delay shift uses only a single synapse, and optimizes the corresponding delay. An early example of delay shift was the Delay Learning Remote Supervised Method (DL-ReSuMe) that showed improved performance compared to just training weights both in terms of accuracy and training speed . Wang et al. further improved upon these results and Shrestha and Orchard extended the approach to enable synaptic delay learning in deep networks. In this paper, we use Dilated Convolutions with Learnable Spacings (DCLS) which, similarly to the approach proposed by Wang et al., convolves spike trains with delay kernels. However, DCLS can be used in deeper architectures and has been shown to be an effective delay learning method on neuromorphic benchmarks . Section title: 1 Introduction Educational score: 4.46795654296875 Domain: biomedical Document type: Study Language: en Methods to decrease the number of parameters in a neural network were first proposed more than 35 years ago and, in recent years, overparametrisation in deep neural networks has become a widely acknowledged problem . The lottery ticket hypothesis proposes that, within an overparameterised dense network, there exist multiple sparse sub-networks with varying performances and, among them, one sub-network stands out as the “winning ticket” that outperforms the others . Sparse neural networks significantly reduce memory usage and energy consumption and are required on many neuromorphic systems, which often have a maximum fan-in per neuron or limited memory available for connectivity . One means of obtaining a sparse network is to train a dense network and remove connections using a process called “pruning” . However, this means that the size of models is still limited by the training cost of the original dense model. In contrast, biological systems dynamically rewire synaptic connections during learning, suggesting that dynamic pruning (also known as structure learning) and rewiring can enhance neural network performance and efficiency. The first algorithm that both disconnected and reconnected neurons during training was DEEP R . The method drops synapses based on their weight changes during learning and replaces them with randomly chosen synapses to maintain a constant number of synapses. In parallel, another dynamic pruning method called sparse evolutionary training (SET) was introduced, relying purely on weight magnitudes to drop connections . Sparse Networks from Scratch (SNFS) used the momentum of each parameter as the criterion for reconnecting neurons . RigL took this one step further and uses gradient information for growing the network. These dynamic pruning methods aim to emulate biological efficiency, potentially offering superior sparsity and accuracy with fewer floating-point operations (FLOPs). According to calculations by Evci et al., at 90% sparsity, RigL requires 1/4 of FLOPs compared to the same size dense model, and DEEP R requires 1/10. With implementations exploiting sparsity, both algorithms can significantly speed up training and inference . Section title: 1 Introduction Educational score: 3.819061517715454 Domain: biomedical Document type: Study Language: en In this paper, we present our analysis of a spiking neural network trained in a supervised fashion on the Heidelberg Digits benchmark dataset , preprocessed as described by Zenke and Vogels . We analyze the learnt parameters of the fully connected network, then train networks with dynamic pruning and fixed sparse connectivity, and conduct the same analysis on these more efficient and biologically more plausible architectures. Section title: 2 Methods Educational score: 2.8635053634643555 Domain: biomedical Document type: Other Language: en We consider an SNN with Leaky-Integrate-and-Fire (LIF) neurons, solved with a linear Euler method, Section title: 2 Methods Educational score: 4.215235710144043 Domain: biomedical Document type: Study Language: en where, u i ( l ) [ t ] is the membrane potential of the i -th neuron in layer l at time step t , τ = 10.05 is the membrane time constant and I i ( l ) ( t ) is the input current. S i ( l ) is the spike train emitted by neuron i , ϑ denotes the firing threshold and Θ the Heaviside function. For our numerical experiments, we used a timestep of Δ t = 1. Section title: 2 Methods Educational score: 3.789573907852173 Domain: biomedical Document type: Study Language: en Because Θ is non-differentiable, we replace it with a arctan surrogate gradient during the backward pass of our training . To implement the synaptic delay training, the input current I i ( l ) ( t ) is calculated using DCLS , i.e., convolving the spike train S j ( l - 1 ) from layer l −1 with the 1D kernel Section title: 2 Methods Educational score: 4.147134780883789 Domain: biomedical Document type: Study Language: en where T d = 25 is the maximum delay, d i j ( l ) is the synaptic delay from neuron j to neuron i in layer l , c is a normalization term so that ∑ n = 0 T d k i j ( l ) [ n ] = w i j ( l ) and σ = 12.5 is the standard deviation of the delay kernel, which is decreased during training. As the kernel k i j ( l ) slides through the spike train S j ( l - 1 ) at each timestep t the kernel will have access to spikes in the range of { t − T d , …, t }. The method essentially creates a temporal convolutional kernel, where the position of weights in the kernel corresponds to the synaptic delay. These weights can then be learned as normal, providing a framework in which weights and delays can be optimized together. For more details, we refer the reader to the original publication . Section title: 2 Methods Educational score: 4.063044548034668 Domain: biomedical Document type: Study Language: en For dynamic pruning, we combined two methods: DEEP R for dropping connections and RigL for introducing them. In DEEP R the synaptic weights are defined as w i j l = s i j ( l ) max ( θ i j ( l ) , 0 ) , where s i j ( l ) is a constant sign value, and θ i j ( l ) is the parameter trained with gradient descent. If θ i j ( l ) is not positive, the connection is considered dormant. We use L 1 regularization to encourage pruning of unnecessary connections. Although the original DEEP R method adds noise to the gradients to induce stochasticity in ANNs, following Bellec et al. , we omitted this in our experiments. DEEP R maintains a fixed number of synapses by randomly reactivating synapses throughout training. Section title: 2 Methods Educational score: 2.7269320487976074 Domain: biomedical Document type: Other Language: en RigL introduces synapses based on gradient analysis rather than randomly. In each iteration, the algorithm selects the k strongest negative gradients from the inactive connections: Section title: 2 Methods Educational score: 2.2751801013946533 Domain: biomedical Document type: Other Language: en Since we make the assumption that the weight of active connections is positive, the original RigL method needs to be modified slightly. Instead of picking synapses to introduce based on the absolute gradient value, we simply pick based on the strongest negative gradient values, since a positive gradient implies that gradient descent wants to keep the connection inactive. Section title: 2 Methods Educational score: 3.3299810886383057 Domain: biomedical Document type: Study Language: en Most neural network models do not adhere to Dale's law, which states that neurons are either exclusively excitatory or inhibitory. Computationally, this means that the signs for the outgoing weights from each presynaptic neuron are the same. Since with DEEP R we have to generate the sign matrix before training, we can conveniently apply this constraint by generating a random sign vector s ∈{−1, +1} and broadcasting it into a matrix. Unless stated otherwise, all of our results apply to networks that adhere to Dale's law. Section title: 2 Methods Educational score: 2.9812276363372803 Domain: biomedical Document type: Study Language: en To measure the spatial autocorrelation of the learned spatio-temporal patterns, we used the method of Moran's I : Section title: 2 Methods Educational score: 4.101090908050537 Domain: biomedical Document type: Study Language: en where N is the number of elements, x are the elements in the pattern, x ¯ is the mean of the elements, w ij are the elements of the spatial weights with zero diagonals, and W is the sum of all w ij . We used the 8 neighborhood case (also known as the Queen's case) for the weights w ij to capture a broad influence. Since the ordering in the spatial dimension is arbitrary (i.e. the ordering of the rows (neurons) can be changed), we took 2000 random row permutations of the matrix and determined the maximum Moran's I across them. With no spatial autocorrelation Moran's I is - 1 N - 1 , meaning that it approaches 0 with increasing N . Moran's I values significantly below - 1 N - 1 indicates negative spatial autocorrelation whereas those significantly above - 1 N - 1 indicate positive spatial autocorrelation. We measure if the distributions of Moran's I values in trained and untrained networks are significantly different using the Mann-Whitney U test. Section title: 2 Methods Educational score: 3.7723066806793213 Domain: biomedical Document type: Study Language: en We extended the PyTorch implementation of DCLS developed by Hammouamri et al. for our experiments. Our architecture consisted of one hidden layer with 256 neurons, dropout layers with a probability of 0.4, batch normalization, and delays in all layers in the range of (0, …, 25) timesteps. We used a voltage sum readout and cross-entropy loss for training and our model was trained with the Adam optimizer, using a OneCycle scheduler for weights and a Cosine Annealing scheduler for delays. Section title: 3.1 Spatio-temporal receptive fields of trained networks Educational score: 4.169267654418945 Domain: biomedical Document type: Study Language: en In our experiments, we trained our models with the best hyperparameters used by Hammouamri et al. for training on the Spiking Heidelberg Digits (SHD) dataset, but instead trained on the Raw Heidelberg Digits dataset . We preprocessed the dataset similarly to Zenke and Vogels , creating Mel spectrograms of shape 40 (input channels) by 80 (timesteps). We performed our experiments on this dataset because it carries complex enough temporal information to benefit from delay learning, but it can be solved with a relatively small network, which makes it better suited for our analysis. The only additional parameter we needed to tune was the L 1 regularization strength. We first trained our model without any sparsity or Dale's law-derived constraints and achieved 97.2%. This is higher than the results reported by Zenke and Vogels (94 ± 2%) – the only other paper running benchmarks on RawHD. However, the goal of this paper is not benchmarking. We then analyzed the learnt spatio-temporal patterns by extracting what we refer to as “spatio-temporal receptive fields” using the process illustrated in Figure 1A . While, in most settings, receptive fields refer to functional attributes of the network , we use a similar concept to analyse the structural attributes of a trained network in terms of the sign and delay of connections. For each hidden neuron, we created a panel with input neurons on the y-axis and delay along the x-axis. Then, for each input neuron, we place one point at the x coordinate corresponding to the learnt delay of its connection to the hidden neuron and colored either blue or red based on the weight's sign. We then summed these panels for each output layer neuron, weighting each by the learned hidden-to-output weight and aligning them on the x-axis according to the learned hidden-to-output delay. These figures allow us to see whether a certain feature for a given class is excited or inhibited and whether it takes effect immediately or with a delay. The results for the dense network before and after training are shown in Figure 1B . Visually, the figure illustrates that spatio-temporal patterns of excitation and inhibition formed after training. Section title: 3.2 Spatio-temporal autocorrelation of learned receptive fields Educational score: 4.174345970153809 Domain: biomedical Document type: Study Language: en We assessed the spatio-temporal autocorrelation of each of the 20 output neuron's trained receptive fields by calculating the maximum Moran's I from 2,000 random row-wise permutations (as the spatial ordering of neurons in our architecture is arbitrary). We repeated our experiment 3 times and created distributions from the 3 × 20 receptive fields for trained and untrained networks . To assess whether there is a meaningful difference in spatio-temporal correlation within receptive fields between trained and untrained networks, we analyzed the distributions of Moran' I values for both network types using a Mann-Whitney U test. This non-parametric test was chosen because it does not assume a specific distribution shape for the data, making it suitable for comparing independent samples with potentially different variances and non-normality. For the dense networks, the Mann-Whitney U test yielded a test statistic of 3, 598.0 and a p -value of approximately 3.88 × 10 −21 . Given this very low p -value, we reject the null hypothesis (H0) that the distributions of Moran's I values in trained and untrained dense networks are identical. This result indicates a highly statistically significant difference between the two distributions with the trained dense networks exhibiting systematically different spatial correlations than the untrained networks. Section title: 3.3 Dynamic pruning Educational score: 4.132364273071289 Domain: biomedical Document type: Study Language: en Next, we trained networks with dynamic pruning—utilizing DEEP R and RigL to enforce a fixed level of 87.5% sparsity and Dale's law by making all of each neuron's outgoing connections have the same sign. While, visually, the emergence of grouping is not as obvious as it was in the dense networks , calculating Moran's I and performing a similar Mann-Whitney U test produced a test statistic of 3, 169.0 and a p-value of approximately 6.69 × 10 −13 . Although this p-value is higher than that observed for the dense network, it still provides strong evidence to reject the null hypothesis, indicating a statistically significant difference between the trained and untrained distributions for the sparse network. Section title: 3.3 Dynamic pruning Educational score: 4.115395545959473 Domain: biomedical Document type: Study Language: en These findings suggest that training introduces structural features within the network's receptive fields, contributing to increased spatial correlation that is absent in untrained networks. While the effect is more pronounced in dense networks, the presence of a significant difference in the sparse network, despite its high level of sparsity, highlights that spatial correlations remain an important outcome of the training process. This may imply that the network's learning captures and reinforces spatial dependencies critical to its task, as reflected in the elevated Moran's I values in trained networks. Section title: 3.4 Ablation study Educational score: 4.104230880737305 Domain: biomedical Document type: Study Language: en In this section, we analyse the combined and separate usefulness of dynamic pruning (i.e. structure learning) and delay learning. We defined four models, one where the structure is learnt, one where the synaptic delays are learnt, one where both are learnt and one where neither are (the synaptic weights are still trained in all cases). See Figure 2 for illustration. For the simulations with fixed connectivity, we omitted Dale's law, since for the models with fixed connectivity, poor initialization could impose significant constraints on the network. Starting with a 50% sparsity, we progressively halved the number of connections in a sequence of experiments. The effectiveness of DEEP R and RigL seems highly dependent on initialization, so we ran three repeats of each configuration and reported the average classification accuracy. Figure 3 demonstrates that dynamic pruning is highly effective, especially as sparsity increases. While learning delays is beneficial when the structure is fixed, its benefits are less obvious when the structure is learnt. Overall, dynamic pruning seems vital for maintaining high performance in sparse networks and we would argue that, when structure is learned, delay learning might not be necessary. Section title: 4 Discussion Educational score: 4.150825500488281 Domain: biomedical Document type: Study Language: en SNNs have a strong potential for spatio-temporal processing and synaptic delays only enhance this. Our study demonstrates a new approach for the visual analysis of networks with delay learning, revealing that functional spatio-temporal patterns emerge in both dense and sparse networks. These patterns appear more in networks where the framework allows for the optimization of temporal parameters through gradients or structural learning. We compared the Moran's I distributions of models trained with and without delay learning and dynamic pruning. The mean Moran's I value of the model with fixed delays and structure was much lower (0.027 compared to 0.064) and the distributions were significantly different (Mann-Whitney U test statistic of 166.5 and p-value of approximately 9.97 × 10 −18 ). At the same time the classification performance is less , suggesting that emerging spatio-temporal structure and classification success correlate. Section title: 4 Discussion Educational score: 4.2035417556762695 Domain: biomedical Document type: Study Language: en While delay learning seems useful with sparse connectivity, we found that learning the structure is more important. In fact, when the structure was learnt, delay learning appeared to bring little benefit. This may be because gradient descent struggles with simultaneously learning both parameters; if a newly connected synapse has a non-optimized delay, it might be immediately deactivated again. However, our experiments with using RigL for removing connections (which does not happen every epoch like DEEP R) did not show significant benefits, challenging this theory. From another perspective, structure learning with fixed delays is a form of delay learning since, if a synapse has an ineffective delay, the network can adapt by introducing a more effective synapse with a new random delay. This method closely resembles “delay select” SNNs . However, for this method to work in the fully connected setting, several connections are required between each pre and postsynaptic neuron, with the number increasing as the delay distribution widens. Ergo, from the point of view of efficiency, we have an argument for delay learning methods such as the one based on DCLS, especially in the fully connected setting. However, we argue that a good test for the usefulness of a given delay learning method is whether, in a sparse network, it performs better than simply replacing synapses with new ones that have a fixed random delay. In our experiments, we relied on gradient information to reconnect weights, but this only yielded minor improvements over random reconnection (standard DEEP R). This implies that, in our simulations, randomly sampling a delay value was just as effective as adjusting delays using gradient descent. While both in our experiments and the ones run by Hammouamri et al. , delay learning does improve network performance, perhaps the precision that delay gradient information provides is not necessary and slows down learning performance. Section title: 4 Discussion Educational score: 1.4443202018737793 Domain: other Document type: Other Language: en In conclusion, our results show that sparse network architectures can be efficient for machine learning tasks. Our findings pave the way for future research into the optimization of SNNs for various applications, particularly those at the edge that have strong memory and computational constraints. | Other | biomedical | en | 0.999995 |
PMC11695356 | Section title: Introduction Educational score: 4.740034103393555 Domain: biomedical Document type: Review Language: en Mycoplasma hyopneumoniae ( M. hyopneumoniae ) is considered one of the smallest known bacteria and lacks a cell wall, with a genome size of approximately 0.86–0.96 Mb, including the P36 gene which has been shown to be highly homologous . The P36 gene also has high specificity for differentiating M. hyopneumoniae from other microorganisms . M. hyopneumoniae serves as the primary pathogen responsible for enzootic pneumonia in swine. Additionally, it plays a crucial role as one of the key agents in the Porcine Respiratory Disease Complex . M. hyopneumoniae is widespread worldwide and detection rates have risen dramatically since 2018, causing major economic losses to the swine industry . The increase in prevalence can be ascribed to the heightened focus on control measures for African swine fever for its initial outbreak in China in 2018 . Furthermore, vaccination against M. hyopneumoniae is not compulsory and vaccines are not widely utilized on farms . Antibiotic treatment (tetracyclines, macrolides and lincosamides) can alleviate clinical symptoms and reduce bacterial load in infected individuals, but do not eliminate the infection so that a risk of reinfection . M. hyopneumoniae , which can reside in the tonsils and nasal cavities of healthy pigs, can also induce disease when isolated from apparently unaffected animals . Therefore, rapid diagnosis is crucial to prevent and control an epidemic of M. hyopneumoniae . Currently, the main detection methods regarding M. hyopneumoniae in swine include isolation culture, serologic assays and nucleic acid assays. Although identification of M. hyopneumoniae through bacterial culture is regarded as the “gold standard”, isolation is difficult due to its fastidious growth requirements and slow growth rate . Many serological assays for M. hyopneumoniae have been developed to monitor the health status of pig herds , but most serological assays cannot distinguish between natural infections and vaccinated animals, leading to potential false-positive results . Nucleic acid assays for M. hyopneumoniae , such as polymerase chain reaction (PCR) , nested PCR (nPCR) and real-time quantitative PCR (qPCR) , have been used for detection in the laboratory. Moreover, the PCR, nPCR and qPCR detection methods for M. hyopneumoniae were also adopted in the Chinese national standard , Chinese agricultural industry standard and Chinese entry–exit inspection and quarantine industry standard . However, relatively expensive instruments such as PCR machines and electrophoresis apparatus are necessary to perform these tests, which restrict their use in many small front-line laboratories. Techniques such as recombinase polymerase amplification , recombinase-aided amplification (RAA) and loop-mediated isothermal amplification have been developed as alternative approaches. Compared to traditional PCR technology, isothermal amplification does not need expensive experimental equipment and consumables, and therefore has a wider application range and easier operation. However loop-mediated isothermal amplification requires more than four specific primers and complicated primer design. In addition RAA and recombinase polymerase amplification are susceptible to non-specific amplification at low template concentrations or in the absence of a template because they operate under isothermal conditions. Section title: Introduction Educational score: 4.266134262084961 Domain: biomedical Document type: Study Language: en In recent years, the CRISPR/Cas system, combined with the shorter CRISPR RNA (crRNA) and Cas proteins , has emerged as a potential tool for rapid, sensitive and highly-specific molecular diagnosis , which relies on the cleavage preferences of Cas12 or Cas13 in a nonspecific way after binding to a specific target DNA or RNA through crRNA . Among those, the Cas12a-based system using a single-stranded DNA probe is more suitable for the detection of bacterial pathogens . The combination of the CRISPR/Cas system with isothermal amplification can be easily used for the detection of nucleic acids, such as SHERLOCK , DETECTR and HOLMES , and the result can be combined with fluorescent probes or immunochromatography technology to express results. CRISPR/Cas12a-based detection has been successfully applied to detect porcine respiratory bacterial pathogens, such as Actinobacillus pleuropneumoniae ( A. pleuropneumoniae ) , Streptococcus suis ( S. suis ) , Haemophilus parasuis ( H. parasuis ) and Pasteurella multocida ( P. multocida ) . Section title: Introduction Educational score: 4.178858757019043 Domain: biomedical Document type: Study Language: en The P36 gene of M. hyopneumoniae is suitable for designing primer and probe to enhance detection accuracy and sensitivity . It enables rapid and precise identification of M. hyopneumoniae in clinical samples for early diagnosis and treatment. The P36 gene of M. hyopneumoniae has previously been reported in PCR and loop-mediated isothermal amplification (LAMP) detection methods . In this study, primers for RAA and crRNA in vitro transcription templates based on the P36 gene of M. hyopneumoniae were designed. A rapid detection platform for M. hyopneumoniae based on RAA with the CRISPR/Cas12a system was successfully developed, which was not only highly sensitive and specific, but even suitable for detection at front-line sites. It provides a highly-convenient method of microbial quality control of specific pathogen-free pigs and of field diagnosis and detection in the swine industry. Section title: Pathogenic nucleic acids and clinical samples Educational score: 2.120129108428955 Domain: biomedical Document type: Study Language: en Genomic (DNA or cDNA) of M. hyopneumoniae , Mycoplasma hyorhinis ( M. hyorhinis ), A. pleuropneumoniae , H. parasuis , S. suis , P. multocida , porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), Mycoplasma capricolum ( M. capricolum ), Mycoplasma synoviae ( M. synoviae ) and Mycoplasma gallisepticum ( M. gallisepticum ) were stored in the State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. In addition, we gratefully acknowledge the contribution of 51 lung tissue samples and 25 nasal swab samples from pigs, kindly provided by other laboratories at the Harbin Veterinary Research Institute. The Animal Ethics Committee of Harbin Veterinary Research Institute did not require ethical review or approval for this study. Section title: Design and synthesis of crRNA Educational score: 4.112560272216797 Domain: biomedical Document type: Study Language: en The P36 gene of M. hyopneumoniae has been shown to be highly homologous , so we selected it as a test gene and used fragments of the conserved region for the design of crRNA. Four DNA templates for crRNA synthesis were designed using the CHOPCHOP online tool . The four DNA templates were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The four DNA templates underwent an annealing heat treatment. Subsequently, in vitro transcription was performed by incubating at 37°C for 16 hours, following the instructions provided by the HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs, Beverly, MA, USA). The final transcription products were treated with DNase I (New England Biolabs) to remove residual DNA template and purified using the Monarch RNA Cleanup Kit Protocol Card (New England Biolabs). The concentration of the final crRNA was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and stored at -80°C for future use. The sequences of all oligonucleotides synthesized in this study are presented in Table 1 . Section title: Preparation of standard plasmid Educational score: 4.138271331787109 Domain: biomedical Document type: Study Language: en Plasmid was constructed by PCR amplification of genomic DNA using the primer pairs Mhp P36 F1 and R1 ( Table 1 ) designed in the conserved regions of the P36 gene. The PCR products were purified using a gel extraction kit (Omega, Norcross, GA, USA) according to the manufacturer’s instructions. The purified fragments were ligated into the pMD-18T vector (Takara Bio Inc., Dalian, China) and transformed into DH5α competent cells (Takara Bio Inc.) for overnight culture, resulting in plasmid isolation. The concentration of the plasmid was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific), and the standard plasmid copy number was calculated using the following formula: copies/μL = (A260 (ng/μL) × 10 −9 × 6.02 × 10 23 )/(DNA length × 650). A ten-fold dilution series ranging from 1 × 10 8 to 1 × 10 -1 copies/μL of the standard plasmid was prepared using EASY Dilution (Takara Bio Inc.) and stored at -20°C for subsequent experiments. Section title: RAA amplification and primer design Educational score: 4.1183037757873535 Domain: biomedical Document type: Study Language: en Standard RAA reactions were conducted according to the instructions of the Basic Nucleic Acid Amplification Kit (ZC Bio-Sci&Tech Co. Ltd, Hangzhou, China). Each reaction mixture contained 25 µL of A Buffer, 2 µL each of forward and reverse primers (10 µM), 5 µL of genomic DNA, 2.5 µL of B buffer, and 13.5 µL of nuclease-free H 2 O. The mixture was incubated in a water bath at 39°C for 30 min. Six primer pairs, denoted as RAA-F1/R1, RAA-F2/R2, RAA-F3/R3, RAA-F4/R3, RAA-F4/R4, and RAA-F4/R2 ( Table 1 ), were generated using Primer 5.0 software. These primers were designed based on sequences flanking the crRNA probe region, ensuring no overlap with the crRNA region. The design parameters for these primers include a product length range of 150-250 bp, primer length between 30 and 35 bp, and GC content ranging from 30% to 70%. Section title: Screening of RAA primers and optimization of amplification time Educational score: 4.087212085723877 Domain: biomedical Document type: Study Language: en Six primer pairs (RAA-F1/R1, RAA-F2/R2, RAA-F3/R3, RAA-F4/R3, RAA-F4/R4 and RAA-F4/R2) ( Table 1 ) were used to perform RAA reactions separately serving standard plasmids of 1 × 10 3 copies/µL as a template. Primer pairs with higher amplification efficiency and specificity were conceived as candidates. Then, the amplification template was switched from standard plasmid to genome, and following the above procedure, the primer pair with the highest specificity and amplification efficiency among the candidate primer pairs was selected for subsequent experiments. Furthermore, we explored the optimal RAA amplification time. Reaction times of 15 min, 20 min, 25 min and 30 min were tested, with each experiment repeated three times. By comprehensively analyzing the fluorescence generation time, fluorescence curve trends, and fluorescence intensity influenced by the five different RAA reaction times, the optimal RAA reaction time was determined. Section title: Establishment and optimization of the CRISPR/Cas12a reaction system Educational score: 4.123604774475098 Domain: biomedical Document type: Study Language: en The unoptimized reaction system (50 μL) consisted of the following mixture: 1 μL LbCas12a (5 µM) (Bio-lifesci Co., Ltd., Guangzhou, China), 5 μL 10× buffer, 2 μL crRNA (5 µM) ( in vitro synthesis), 2 μL ssDNA reporter probe (10 µM) (Sangon Biotech Co., Ltd., Shanghai, China), 3 μL template, with the remainder made up with nuclease-free H 2 O. The mixture was incubated for 25 min at 37°C in a water bath and a microplate reader was used for detection (PerkinElmer, Waltham, MA, USA). The crRNA was screened using this reaction system and conditions and the most suitable crRNA was identified based on the final fluorescence intensity and reaction efficiency for subsequent experiments. In the assay, the final concentrations of LbCas12a, crRNA and ssDNA were optimized using the checkerboard method to optimize concentrations of LbCas12a and crRNA by fixing other components, which was set up with 30, 60, 120, 180 and 240 nM of crRNA and 20, 40, 60, 80, 100 and 200 nM of LbCas12a. ssDNA was optimized by fixing other components and adjusting the concentration to 100, 200, 300, 400 and 500 nM. Experimental data measured using a microplate reader (PerkinElmer) were processed to determine the optimal concentrations of LbCas12a, crRNA and ssDNA for the final reaction system. Section title: Readout formats Educational score: 4.310294151306152 Domain: biomedical Document type: Study Language: en The results can be expressed in terms of fluorescence using a microplate reader and visualized under blue light or through lateral flow assay (LFA). For fluorescence detection, the ssDNA reporter is labeled with FAM and BHQ1 in the fluorescent and quencher groups, respectively. We use a microplate reader (PerkinElmer) with excitation and emission wavelengths set at 494 nm and 518 nm to measure the fluorescence intensity. Additionally, a blue light transilluminator can be used to observe fluorescence, where the presence of green fluorescence serves as an indicator of the target DNA’s existence. For LFA detection, the ssDNA reporter is labeled with FAM and biotin at the 5’ and 3’ ends, respectively. Following the instructions, we place the lateral flow assay strip (GenDx Biotech Co., Ltd., Suzhou, China) into the incubation product, yielding results within 2 min. The lateral flow strip comprises three distinct sections: the sample zone, the control line and the test line. The sample zone is coated with gold-labeled anti-FAM antibodies. The control line area is covered with streptavidin, which captures uncleaved ssDNA reporter molecules labeled with biotin. The test line area is coated with anti-mouse antibodies designed to detect the cleaved ssDNA reporter labeled with gold nanoparticles. For negative samples, the gold-labeled anti-biotin antibodies fully bind to the abundant biotin-labeled ssDNA reporter, resulting in complexes that are intercepted by anti-FAM antibodies at the control line. Conversely, for positive samples, the ssDNA reporter is cleaved, leading to the accumulation of gold-labeled anti-biotin antibody at the test line. The colorimetric process should ideally be completed within 4 min. Positive results are indicated by the presence of a red control line and test line, or only a red test line. In contrast, results are considered negative when only the control line shows a red color, and invalid when no lines are displayed. Section title: Specificity and sensitivity of the RAA-CRISPR/Cas12a fluorescence detection method Educational score: 4.026836395263672 Domain: biomedical Document type: Study Language: en In order to investigate the specificity of the RAA-CRISPR/Cas12a fluorescence detection method, genome DNA of M. hyopneumoniae , M. hyorhinis , A. pleuropneumoniae , H. parasuis , S. suis , P. multocida , PCV2, PRV, M. capricolum , M. synoviae and M. gallisepticum and genome cDNA of PRRSV and SIV were detected by the final reaction system. Meanwhile, nuclease-free H 2 O was used as a negative control. Section title: Specificity and sensitivity of the RAA-CRISPR/Cas12a fluorescence detection method Educational score: 4.138217449188232 Domain: biomedical Document type: Study Language: en To evaluate the sensitivity of the RAA-CRISPR/Cas12a fluorescence detection method, 10-fold dilutions of standard plasmids ranging from 1 × 10 4 to 1 × 10 -1 copies/μL and 5 copies/μL of standard plasmid were used as templates to determine the limit of detection (LoD) for the RAA-CRISPR/Cas12a-fluorescence. Additionally, using the 10-fold dilution of 1 × 10 8 –1 × 10 -1 copies/μL of the standard plasmid as templates, we tested the sensitivity of qPCR according to the recommended Chinese entry–exit inspection and quarantine industry standard . The qPCR reaction mixture (20 μL) contained 10 μL Premix ExTaq (probe qPCR) (2×), 0.4 μL of each Mhp-SN-F/R primer (10 µM), 0.8 μL Mhp-SN-probe (10 µM), 0.4 μL Rox Reference DyeII, 5 μL template and 3 μL nuclease-free H 2 O. The cycling conditions were set as follows: initial denaturation at 95°C for 3 min, subsequently proceeding to 40 cycles, each consisting of denaturation at 95°C for 15 seconds and annealing at 55°C for 45 seconds. Section title: Optimization of the RAA-CRISPR/Cas12a LFA time Educational score: 4.06739616394043 Domain: biomedical Document type: Study Language: en The reaction time of the RAA-CRISPR/Cas12a lateral flow assay was optimized for use in the final reaction system. Four sets with reaction times of 5 min, 10 min, 15 min and 20 min were optimized using 1 × 10 2 copies/µL of standard plasmid as templates, and incubated in a 37°C water bath to determine the minimum reaction time. The optimal reaction time was selected by observing the test line and control line. Section title: Specificity and sensitivity of the RAA-CRISPR/Cas12a LFA Educational score: 3.9879066944122314 Domain: biomedical Document type: Study Language: en In order to evaluate the specificity of the RAA-CRISPR/Cas12a LFA, the thirteen pathogens M. hyopneumoniae , M. hyorhinis , A. pleuropneumoniae , H. parasuis , S. suis , P. multocida , PRRSV, SIV, PCV2, PRV, M. capricolum , M. synoviae and M. gallisepticum were analyzed. Nuclease-free H 2 O was used as a negative control. Section title: Specificity and sensitivity of the RAA-CRISPR/Cas12a LFA Educational score: 4.075686931610107 Domain: biomedical Document type: Study Language: en For sensitivity assessment, 10-fold serial dilutions of 1 × 10 4 –1 × 10 -1 copies/μL and 5 copies/μL of the standard plasmid were used as templates to determine the LoD for the RAA-CRISPR/Cas12a LFA. Section title: Specificity and sensitivity of the RAA-CRISPR/Cas12a LFA Educational score: 3.106862783432007 Domain: biomedical Document type: Study Language: en Specificity and sensitivity of M. hyopneumoniae were determined by observing the control and detection lines of the test strips. Section title: Assays of clinical sample Educational score: 4.136122703552246 Domain: biomedical Document type: Study Language: en Genomic DNA was extracted from 51 lung tissue samples (17 apical lobes, 17 cardiac lobes and 17 diaphragmatic lobes) using a blood/cell/tissue genomic DNA extraction kit (Tiangen Biotech Co. Ltd., Beijing, China) and from 25 nasal swab samples using a bacterial genomic DNA extraction kit (Tiangen Biotech Co. Ltd.). The DNA samples were tested using the final RAA-CRISPR/Cas12a-fluorescence and RAA-CRISPR/Cas12a LFA. To validate the clinical performance, the same samples were analyzed by PCR recommended by the Chinese national standard and qPCR recommended by the Chinese entry–exit inspection and quarantine industry standard . Genomic DNA of M. hyopneumoniae and nuclease-free H 2 O were used as positive and negative controls, respectively. The reaction mixture (25 μL) for PCR contained 12.5 μL 2× Taq PCR StarMix (Dye), 2 μL of each Mhp-GB-F/R primer (10 µM), 3 μL template and 5.5 μL nuclease-free H 2 O. The reaction conditions were set as follows: pre-denaturation at 94°C for 2 min, followed by PCR performed for 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min. Section title: Statistical analysis Educational score: 2.550837755203247 Domain: biomedical Document type: Study Language: en Data analysis and graphic design were processed by GraphPad Prism v.8.1.2 software. Data in the figures are presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) was used for multigroup comparisons. All comparisons were considered statistically significant at P < 0.05 and highly significant at P < 0.001. Section title: Schematic representation of the RAA combined with CRISPR/Cas12a system rapid detection platform Educational score: 4.079038619995117 Domain: biomedical Document type: Study Language: en An overview of the rapid detection platform for M. hyopneumoniae based on RAA-CRISPR/Cas12a is depicted in Figure 1 . First, genomic DNA is extracted from bacterial culture using a bacterial genomic DNA extraction kit. Next, the target DNA is amplified through RAA at 39°C, followed by transfer of the RAA products into the CRISPR/Cas12a reaction system. Finally, the presence of M. hyopneumoniae is determined by visual inspection under blue light, utilization of a lateral flow assay, or fluorescence measured using a microplate reader. The entire process can be completed within one hour. Section title: Screening of RAA primers and optimization of amplification time Educational score: 4.104366302490234 Domain: biomedical Document type: Study Language: en Because the primer sequences are important in RAA amplification, primer screening is necessary. We used standard plasmid and DNA of M. hyopneumoniae as templates to identify the optimal primer pair. Four primer pairs (RAA-F1/R1, RAA-F2/R2, RAA-F4/R4, RAA-F4/R2) were selected using a standard plasmid of 1 × 10 3 copies/µL as a template based on the amplification profile . Then, RAA-F1/R1 was selected for subsequent experiments, as it exhibited the most efficient performance as well as being highly specific when tested using DNA of M. hyopneumoniae as a template . Based on the five sets of reaction times designed for the RAA, the optimal RAA reaction time was determined to be 15 min by considering the fluorescence intensity and background fluorescence values influenced by different reaction durations . Section title: Screening crRNA and optimization of the reaction system Educational score: 4.1057634353637695 Domain: biomedical Document type: Study Language: en Four designed crRNA probes were capable of recognizing distinct 24-nucleotide regions of the P36 gene . Among them, crRNA 1 exhibited the highest fluorescence value and the fastest reaction rate. Therefore, crRNA 1 was selected for use in subsequent experiments. The final optimized reaction system comprised 0.6 μL LbCas12a (final concentration of 60 nM) , 5 μL 10 × buffer, 1.8 μL crRNA (final concentration of 180 nM) , 2 μL ssDNA reporter probe (final concentration of 400 nM) , and 3 μL RAA product, with the volume made up to 50 μL with nuclease-free H 2 O. Section title: Specificity and sensitivity of the RAA-CRISPR/Cas12a fluorescence detection system Educational score: 4.115896224975586 Domain: biomedical Document type: Study Language: en Genomic DNA of M. hyopneumoniae , M. hyorhinis , A. pleuropneumoniae , H. parasuis , S. suis , P. multocida , PCV2, PRV, M. capricolum , M. synoviae and M. gallisepticum and genomic cDNA of PRRSV and SIV as templates were tested to evaluate the specificity of the RAA-CRISPR/Cas12a-fluorescence system. Only DNA of M. hyopneumoniae reacted rapidly with high fluorescence intensity ( P < 0.001) and did not cross-react with other pathogens . The results showed that the RAA-CRISPR/Cas12a fluorescence detection system was highly specific. Section title: Specificity and sensitivity of the RAA-CRISPR/Cas12a fluorescence detection system Educational score: 4.123497009277344 Domain: biomedical Document type: Study Language: en A 10-fold serial dilution series and 5 copies/μL of standard plasmids were used as templates for the RAA-CRISPR/Cas12a-fluorescence. The result showed that the LoD of the fluorescence assay was 1 copy/μL for M. hyopneumoniae . At the same, the qPCR recommended by entry–exit inspection and quarantine industry standard detected 1 × 10 2 copies/μL (CT < 36) . This result indicates that the fluorescence assay is highly sensitive, being 100-fold more sensitive than qPCR. Section title: Optimization of the RAA-CRISPR/Cas12a LFA time Educational score: 4.098753929138184 Domain: biomedical Document type: Study Language: en The reaction time of the RAA-CRISPR/Cas12a lateral flow assay was optimized based on the final reaction system. When 1 × 10 2 copies/µL of standard plasmid was used as a template, the lateral flow strip only displayed the detection line after incubation for 15 min and 20 min , indicating that it takes a minimum of 15 min for the reaction to be complete. Thus the shortest available reaction time for the RAA-CRISPR/Cas12a LFA was 15 min. Section title: Specificity and sensitivity of the RAA-CRISPR/Cas12a LFA Educational score: 4.103583335876465 Domain: biomedical Document type: Study Language: en The specificity of the RAA-CRISPR/Cas12a LFA was evaluated for the thirteen pathogens M. hyopneumoniae , M. hyorhinis , A. pleuropneumoniae , H. parasuis , S. suis , P. multocida , PRRSV, SIV, PCV2, PRV, M. capricolum , M. synoviae and M. gallisepticum based on the final reaction system. In the LFA strips the test line was only visible with M. hyopneumoniae , for the others only the control line appeared . The sensitivity of the RAA-CRISPR/Cas12a LFA was analyzed using a 10-fold serial dilution and 5 copies/μL of standard plasmids. The LoD was 5 copies/μL . Section title: Application in clinical samples Educational score: 4.111305236816406 Domain: biomedical Document type: Study Language: en In order to evaluate the performance of the established detection platform, 51 lung tissues samples and 25 nasal swab samples were tested using the final RAA-CRISPR/Cas12a fluorescence/LFA . The positive rates for 51 lung tissue samples and 25 nasal swab samples were 100% (51/51) and 28% (7/25) in RAA-CRISPR/Cas12a fluorescence . The same results were obtained by RAA-CRISPR/Cas12a LFA . To verify the accuracy of the method, we tested the clinical samples using PCR as recommended by the Chinese national standard and qPCR as recommended by the Chinese entry–exit inspection and quarantine industry standard . The PCR results indicated a positive rate of 96% (49/51) among 51 lung tissue samples, and the nasal swab samples exhibited a 0% (0/25) positive rate among 25 samples ( Table 2 ). Additionally, the 51 lung tissue samples tested by qPCR were 100% (51/51) positive, meanwhile, the nasal swab samples showed a 16% (4/25) positive rate among the 25 samples examined ( Table 2 ). The results obtained using the RAA-CRISPR/Cas12a fluorescence/LFA method were consistent with those obtained from PCR and qPCR . This suggests that the novel approach is not only accurate and reliable but also exhibits a higher sensitivity. Section title: Discussion Educational score: 4.089564800262451 Domain: biomedical Document type: Study Language: en M. hyopneumoniae has a significant economic impact on the swine industry . M. hyopneumoniae infection is widespread globally and prone to facilitating concurrent infections among animals, particularly with other porcine respiratory pathogens . Currently, the molecular diagnostic techniques of PCR, nPCR and qPCR are well established in the literature and in the Chinese national standard , the Chinese agricultural industry standard and the Chinese entry–exit inspection and quarantine industry standard . Although these techniques have been widely validated as useful tools for detecting the disease, they are still not convenient for use in frontline sites due to the requirements for expensive instruments and specialized operating systems. Therefore, we envisaged an urgent need for a method for detection of M. hyopneumoniae based on the combination of RAA and CRISPR/Cas12a which required minimal equipment, and was highly sensitive and capable of rapid detection. Section title: Discussion Educational score: 4.546816349029541 Domain: biomedical Document type: Study Language: en RAA, a novel isothermal nucleic acid amplification technique, enables rapid amplification of DNA or RNA within 30 min at relatively low temperatures (37–42°C) using a simple water bath . This technology relies on three fundamental enzymes: recombinase, single-stranded DNA-binding protein (SSB) and strand-displacing DNA polymerase , which replace the denaturation cycles required in conventional PCR. The CRISPR/Cas system, known for its reliability, sensitivity, and specificity, has emerged as a valuable tool for genomic editing and nucleic acid diagnosis , but the CRISPR/Cas12a system is more suitable for the detection of bacteria. Combining RAA with the CRISPR/Cas12a system , when the target nucleic acid is present, the specific product is amplified by RAA and specifically recognized by Cas12a protein and crRNA complex, and at the same time, the Cas12a trans-cutting activity is activated, which cuts the fluorescent reporter probes and emits fluorescence to judge the detection results . Moreover, a cost analysis comparison of PCR, qPCR, and CRISPR/Cas12a has been reported , which revealed that the CRISPR/Cas12a system is the easiest to operate and the least demanding in terms of instrumentation, while also allowing for high-throughput assays, both in the laboratory and in on-farm settings. Section title: Discussion Educational score: 4.215164661407471 Domain: biomedical Document type: Study Language: en In our study, we confirmed that RAA-F1/R1 and crRNA 1 serve as the most suitable primer pair and crRNA in the conserved M. hyopneumoniae P36 gene. For RAA we performed the amplification at 37°C for 15 min, which is 15 min shorter than the time given in the initial instructions. We presented the RAA-CRISPR/Cas12a system results in two forms, either measuring fluorescence values with a microplate reader or using lateral flow test strips. The RAA-CRISPR/Cas12a fluorescence and RAA-CRISPR/Cas12a LFA both proved to be highly specific for M. hyopneumoniae and showed no cross-reactivity with other pathogens ( M. hyorhinis , A. pleuropneumoniae , H. parasuis , S. suis , P. multocida , PRRSV, SIV, PCV2, PRV, M. capricolum , M. synoviae and M. gallisepticum ). The sensitivity test revealed a LoD of 1 copy/μL and 5 copies/μL of the RAA-CRISPR/Cas12a-fluorescence assay and RAA-CRISPR/Cas12a- LFA, respectively. Both of these methods can serve as the qualitative test for samples, rather than quantitative analysis. Furthermore, we tested 51 lung tissue samples and 25 nasal swab samples with the RAA-CRISPR/Cas12a system to verify its practicality and usefulness in clinical samples. Fifty-one (100%) positive lung tissue samples and seven (28%) positive nasal swab samples were detected by the RAA-CRISPR/Cas12a fluorescence and RAA-CRISPR/Cas12a LFA, respectively. It can be seen that M. hyopneumoniae infection in pigs should not be ignored and should be detected in time to prevent the spread of the infection. Meanwhile, we also tested the 51 lung tissue samples and 25 nasal swab samples by the PCR method recommended by the Chinese national standard and by the qPCR method recommended by the Chinese entry–exit inspection and quarantine industry standard to verify the accuracy of the RAA-CRISPR/Cas12a system. Forty-nine (96%) of the 51 lung tissue samples and 0 (0%) of the 25 nasal swab samples were detected by PCR. The qPCR testing revealed that 51 (100%) of the 51 lung tissue samples were positive, whereas 4 (16%) of the 25 nasal swab samples tested positive. The results showed consistency rates of 100% between the three methods. Above all, compared with PCR and qPCR, the RAA-CRISPR/Cas12a system is not only more sensitive, but also significantly more stable. Section title: Conclusion Educational score: 4.083616256713867 Domain: biomedical Document type: Study Language: en In conclusion, RAA-CRISPR/Cas12a was established and used as a rapid, highly specific, cost-effective diagnostic and portable detection method for M. hyopneumoniae . The method can be used in a high-throughput mode in swine fields or microbiology diagnostic laboratories, with results obtained in less than one hour. It provides an effective means for field diagnosis of M. hyopneumoniae and microbiological quality control of experimental swine. | Review | biomedical | en | 0.999998 |
PMC11695359 | Section title: Introduction Educational score: 4.418197154998779 Domain: biomedical Document type: Review Language: en Rituximab, a chimeric monoclonal antibody, is directed against the CD20 antigen expressed on B cells and most of their precursors. It was the first ever approved therapeutic antibody and its initial label was the treatment of Non-Hodgkin Lymphoma with a recommended dose of 375 mg/m 2 once weekly for 4 weeks ( 1 ). Meanwhile, it has become standard of care (mostly as part of an immune-chemotherapy) in various B-cell malignancies, such as diffuse large B-cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, and mantle cell lymphoma ( 2 ). Rituximab depletes CD20 + cells by either triggering complement-dependent cytotoxicity or initiating antibody-dependent cellular cytotoxicity ( 3–5 ). Beside its therapeutic effects in B-cell malignancies, rituximab also produces immunomodulatory effects, which led to its application in various autoimmune diseases ( 6–10 ). In rheumatoid arthritis the approved dose is 1,000 mg on day 1 and day 15 ( 7 ), while in ANCA-vasculitis 375 mg/m 2 weekly for 4 weeks or 1,000 mg on day 1 and 15 are recommended ( 6 ). Rituximab is also approved in pemphigus vulgaris, when clinical remission is not achieved with systemic corticosteroids or other immunosuppressive drugs (rituximab 2 × 1,000 mg 2 weeks apart or 375 mg/m 2 weekly x4) ( 8–10 ). Moreover, rituximab is frequently used “off-label” in other autoimmune diseases such as autoimmune hemolytic anemia (AIHA) ( 11 ), thrombotic thrombocytopenic purpura ( 12 ), immune thrombocytopenia ( 13 ), or multiple sclerosis, typically with four doses of 375 mg/m 2 per week ( 14 ). Section title: Introduction Educational score: 3.9987881183624268 Domain: biomedical Document type: Study Language: en Although, the approval for rituximab dates back decades ( 1 ) and the pharmacokinetics (PK) of rituximab have been analyzed in numerous studies, only a single dose finding study was performed in 15 patients with relapsed low-grade B cell lymphoma ( 15 , 16 ). However, there are no formal dose-finding studies for autoimmune diseases ( 15–26 ). This is especially interesting because B-cell malignancies are associated with a much higher antigen burden compared with autoimmune diseases, which may necessitate much higher and more frequent dosing in malignant diseases ( 18 ). Section title: Introduction Educational score: 3.5546326637268066 Domain: biomedical Document type: Study Language: en Several clinical studies already investigated alternative dosing regimens (like 200 mg, 250 mg/m 2 or 500 mg) and overall showed comparable efficacy to standard doses, for instance, in cryoglobulinemia vasculitis or rheumatoid arthritis ( 27 , 28 ). Section title: Introduction Educational score: 3.9040095806121826 Domain: biomedical Document type: Review Language: en In AIHA, autoantibodies directed against erythrocyte surface molecules lead to the destruction of erythrocytes and consecutively to anemia. According to the First International Consensus Meeting, corticosteroids remain first line therapy in AIHA adults ( 29 ), but targeting CD20 + cells by adding rituximab might contribute to a decrease in erythrocyte antibody production and therefore is recommended in severe cases ( 29 , 30 ). Section title: Introduction Educational score: 3.8524367809295654 Domain: biomedical Document type: Study Language: en In former studies ( 31 , 32 ) patients with warm AIHA or with cold agglutinin disease (CAD) received a fixed dose of 100 mg rituximab once a week over 4 weeks with response rates of about 90%. However, despite these studies proving efficacy of lower doses, the investigated dosing regimens were neither based on traditional dose finding studies nor derived from pharmacological parameters. Section title: Hypothesis Educational score: 4.22603702545166 Domain: biomedical Document type: Study Language: en In an earlier trial that included healthy volunteers, the half-maximal effective dose (ED50) of rituximab was approximately 0.1–0.3 mg/m 2 ( 33 ). The half-maximal effective dose is defined as the dose at which a drug reaches 50% of its effect. This parameter is tightly connected to the EC50, which describes the concentration at which 50% of the response of a drug is achieved. These parameters relate to the steep part of the dose–response curve and are used to quantify and/or compare the potency of drugs, but may also be used to describe interindividual variability ( 34 ). At a dose of 1 mg/m 2 rituximab reduced CD20 + cell counts transiently by 97% (the approximate ED95) ( 33 ). Based on these data, we estimated the EC95 to be approximately 600–700 ng/mL. The ED95 and the EC95 correspond to the dose and concentration at which a drug achieves 95% of its intended effect. These parameters, alongside other pharmacological parameters such as absorption or elimination, are important for dose finding and to develop therapeutic regimens. In an in vitro study the half-maximal effective concentration (EC50) of rituximab for depletion of human B cells was found to be approximately 1 μg/mL ( 35 ). Section title: Hypothesis Educational score: 2.7963287830352783 Domain: biomedical Document type: Study Language: en In sum, approved doses exceed these concentrations several hundred-fold supporting the hypothesis that much lower doses may be equally effective ( 15 ). Section title: Hypothesis Educational score: 4.088369846343994 Domain: biomedical Document type: Study Language: en In this study we set out to investigate several dosing regimens that were calculated using these parameters in addition to the well-known terminal elimination half-life of rituximab of approximately 21 days in autoimmune diseases ( 36 ) and tested their effects in patients with AIHA. We hypothesized that in non-malignant diseases a long-lasting and effective suppression of CD20 + cells may be possible using much lower doses of rituximab. Section title: Study design Educational score: 4.1190290451049805 Domain: biomedical Document type: Other Language: en This was a phase II, open label, pilot trial to investigate the effects and the safety of very low doses of rituximab in patients with AIHA. We included patients ≥18 years with a diagnosis of AIHA in whom the treating physicians decided to use rituximab. Patients were excluded if they had received rituximab or other CD20-targeting therapies (e.g., ofatumumab, obinutuzumab or ocrelizumab) within the last 12 months and if they had suppressed (non-detectable) CD20 + cell counts at screening. Moreover, patients treated with high doses of corticosteroids (> 100 mg/day), or intravenous immunoglobulins, because of autoimmune-mediated hemolysis, were not eligible. Any concomitant medication was allowed. A detailed list of all in-and exclusion criteria is presented in Supplementary material . The study was designed to determine whether the different dosing regimens may suppress CD20 + cells for the projected treatment period. Depending on the dose group, the active treatment period lasted between 3 months and up to 9 months: four infusions every 3 weeks in the 5 mg/m 2 group, three infusions every 4 weeks in the 20 mg fixed dose group, three infusions every 3 months in the 50 mg and 100 mg dose group. The duration of the active treatment phase was based on observations how long CD20 + cells are fully suppressed after administration of approved doses in autoimmune diseases ( 37 , 38 ). The active treatment phase of the low dose groups (approximately 3 months) was deemed long enough to assess efficacy, but somewhat limited by the number of visits patients had to pay to the study ward. Of note, the study also included the option to continue treatment for up to 2 years (100 mg rituximab every 3 months), if patients clearly benefited from rituximab treatment. Section title: Study design Educational score: 2.421394109725952 Domain: biomedical Document type: Study Language: en The study was conducted at the Department of Clinical Pharmacology, Medical University of Vienna, between 2016 and 2021. All study procedures complied with the principles set forth in the Declaration of Helsinki and the Good Clinical Practics guideline. The Ethics Committee of the Medical University of Vienna approved the study before its initiation . The COVID-19 pandemic had a significant impact on patient recruitment, because (i) there was a strategy to reduce patient contacts within the healthcare system and (ii) physicians were temporarily reluctant to use rituximab given an assumed negative impact on patient outcomes, which was later confirmed ( 39 ). Section title: Study procedures Educational score: 4.026283264160156 Domain: biomedical Document type: Study Language: en At the screening visit all patients underwent a physical examination, an electrocardiogram, a measurement of oral temperature and vital signs, blood and urine collection for clinical laboratory assays. On study days, vital parameters were monitored (heart rate, blood pressure and oxygen saturation) and recorded. A baseline blood sample was drawn including laboratory parameters indicative of hemolysis (haptoglobin, free hemoglobin, LDH, bilirubin, reticulocytes). All patients received premedication of 5 mg levocetirizine perorally, 1,000 mg paracetamol perorally, and 1,000 mL of 0.9% saline solution intravenously at least 30 min before the first rituximab infusion. Premedication at later time points could be omitted, if CD20 + cells were fully suppressed. The respective rituximab dose was diluted to a final volume of 20 mL and infused over 1 h. Blood sampling was performed 2 h after the end of the infusion and vital signs were measured on an hourly basis. The first control visits were conducted 1 and approximately 7 days after infusion and included the collection of adverse events, concomitant medication, as well as blood sampling and the measurement of vital signs. Based on CD20 + cell counts additional control visits could have been performed. Subsequent rituximab infusions (number 2–3 or 4, as applicable) were conducted according to the respective dosing regimen and with similar study procedures. After the active treatment phase control visits were performed approximately every 3 weeks to measure drug concentrations and CD20 + cell recovery. The final examination was performed 2–4 weeks after the last control visit and included blood sampling, measurement of vital signs, a physical examination, and an ECG. Section title: Dosing rationale and regimen Educational score: 4.17323112487793 Domain: biomedical Document type: Study Language: en In a previous clinical trial, healthy volunteers received very low doses of rituximab (0.1, 0.3, and 1 mg/m 2 ) ( 33 ). Infusion of 0.1 mg/m 2 and 0.3 mg/m 2 reduced CD20 + cells transiently by 68 and 74% and infusion of 1 mg/m 2 rituximab depleted CD20 + cells almost completely (~97%). Four weeks after the infusion of 1 mg/m 2 rituximab CD20 + cell counts recovered to approximately 60% ( 33 ). The quantification of rituximab plasma concentration was somewhat complicated by a limited assay sensitivity and target mediated drug disposition that may be especially pronounced for very low doses. We calculated a basic PK model with the following assumptions: theoretical circulating blood volume of 3,000 mL, EC95 of ~600 ng/mL, ED95 of 1 mg/m 2 , ED50 of 0.1 mg/m 2 , and a terminal elimination half-life of ~21 days ( 36 ). The exact model is available as a Supplementary Figure S1 . In short, we expected an infusion of 5 mg/m 2 every 3 weeks (dose group 1), an infusion of 20 mg fixed dose every 4 weeks (dose group 2), an infusion of 50 mg fixed dose every 3 months (dose group 3) to effectively suppress CD20 + cell counts for the entire scheduled period. As a fourth dose group 100 mg every 3 months was amended to the protocol. Section title: Dosing rationale and regimen Educational score: 3.4118645191192627 Domain: biomedical Document type: Study Language: en Doses were escalated, if the suppression of CD20 + cells was not sufficient in all patients of a cohort (defined as a 95% reduction of CD20 + cell counts from baseline). Section title: Endpoints Educational score: 3.9719388484954834 Domain: biomedical Document type: Study Language: en The primary efficacy endpoint was an ≥95% suppression of CD20 + cells during the active treatment phase. Plasma concentrations of rituximab were quantified and pharmacodynamic endpoints included hemoglobin concentrations, and signs of hemolysis. Due to sparse blood sampling a full analysis of pharmacokinetics was not performed. However, we analyzed the pharmacokinetic/pharmacodynamic relationship in an exploratory manner. Section title: Endpoints Educational score: 3.6007676124572754 Domain: biomedical Document type: Study Language: en Plasma rituximab concentrations were quantified by enzyme-linked immunosorbent assay (VelaLabs GmbH, Vienna, Austria). CD20 + cell counts were determined by flow cytometric analysis (Department of Laboratory Medicine, Medical University of Vienna, Austria). Section title: Endpoints Educational score: 4.0017991065979 Domain: biomedical Document type: Study Language: en Hemolysis-specific laboratory parameters included the following: hemoglobin concentration (local reference range 12–16 g/dL for females, 13.5–18 g/dL for male), reticulocyte counts (32–110 G/L), LDH (< 250 U/L), total bilirubin concentration (0.0–1.2 mg/dL) and haptoglobin concentration (30–200 mg/dL). Safety endpoints included vital signs, adverse events, and safety laboratory parameters. Section title: Statistical analysis Educational score: 3.8766276836395264 Domain: biomedical Document type: Study Language: en Demographics and baseline data are presented by descriptive statistics (mean, standard deviation). The primary endpoint was a continuous ≥95% suppression of CD20 + cells in between dosing intervals. An exact confidence interval with a one-sided nominal coverage probability of 90% for the true rate of responders was calculated. Endpoints are presented descriptively. Section title: Statistical analysis Educational score: 4.132658004760742 Domain: biomedical Document type: Study Language: en In the forementioned trial ( 33 ), 16 healthy volunteers were enrolled. Subjects received 0.1 mg/m 2 (4 subjects), 0.3 mg/m 2 (4 subjects) or 1 mg/m 2 (8 subjects) in a dose escalating manner. CD20 + cell counts showed a large variability with coefficients of variation of approximately 20–60%. The initial depletion of circulating B-lymphocytes was ≥95% after 1 mg/m 2 rituximab infusion ( 33 ). Based on these results, strong treatment effects were expected in patients. For CD20 + cell numbers below the lower limit of quantification we imputed a CD20 + cell count of “0” and assumed treatment success. Treatment success was defined as a ≥95% reduction of CD20 + cells during the entire scheduled period. We desired a 100% response rate, which would result in the lower bound of a one-sided 90% confidence interval of 0.56, 0.68, 0.75 for the sample sizes n = 4, n = 6 or n = 8, respectively, to achieve sufficient evidence for the efficacy of very low-dose rituximab. In case a dose level was found inefficacious, the dose was escalated. We aimed for a minimum sample size of n = 3 per dose group. If the dose was inefficacious, we escalated the dose. However, if a dose effectively reduced CD20 + cell counts according to the defined criteria, more patients were to be enrolled in this dosing cohort. However, due to the early termination of the trial and, because the tested rituximab regimens were found inefficacious, the statistical analysis was done in a descriptive manner only. Section title: Statistical analysis Educational score: 1.3201655149459839 Domain: biomedical Document type: Other Language: en Figures were illustrated with graphpad prism 10. MS excel, and SPSS were used for statistical calculations. Section title: Results Educational score: 2.991180658340454 Domain: biomedical Document type: Study Language: en A total of 14 patients were screened between 2016 and 2021. There were two screening failures (suppressed CD20 + cells in one patient, treatment with immunoglobulins in another patient). Ten patients completed the study, while two patients dropped out . Two patients suffered from warm AIHA, while seven patients suffered from cold agglutinin’s disease and one patient suffered from a mixed-type AIHA with autoantibodies compatible with cold agglutinin’s disease and warm AIHA. The study was terminated early due to slow recruitment of patients, especially during the COVID-19 pandemic. Section title: Results Educational score: 2.7771530151367188 Domain: biomedical Document type: Study Language: en The mean age was 68 years (±10 standard deviation) and the mean weight was 71 kg (± 13). Six female and four male patients completed the study. One patient received rituximab previously for treatment of autoimmune hemolytic anemia more than 12 months ago ( Table 1 ). Section title: Results Educational score: 4.074708938598633 Domain: biomedical Document type: Study Language: en In the first cohort three patients received 5 mg/m 2 every 3 weeks, two patients with CAD and one with mixed AIHA. In one patient with CAD the infusion of 5 mg/m 2 immediately depleted all CD20 + cells. In one patient CD20 + cells were not successfully depleted . In line with these results, rituximab concentrations remained well below 1 μg/mL at all time-points in this subject. Section title: Results Educational score: 3.795870304107666 Domain: biomedical Document type: Study Language: en In the patient with mixed-type AIHA CD20 + cells recovered 1 week after the first infusion (14 CD20 + cells/μL, 0.1 μg/mL rituximab). However, in this subject CD20 + cells were successfully depleted after the second infusion and remained suppressed for the scheduled period. In another patient CD20 + cells were successfully depleted for the whole scheduled period. Section title: Results Educational score: 2.0275375843048096 Domain: biomedical Document type: Other Language: en Based on these results, this dose level was considered partially ineffective, and the dose was escalated. Section title: Results Educational score: 3.3969533443450928 Domain: biomedical Document type: Study Language: en Three patients received 20 mg fixed dose every 4 weeks , two patients with CAD and one with wAIHA. Rituximab infusion successfully depleted CD20 + cells for over 24 h in all three patients. Section title: Results Educational score: 4.095067977905273 Domain: biomedical Document type: Study Language: en In one patient with CAD CD20 + cells remained successfully depleted over the whole study period (114 days = 3 months and 25 days). In the other patient with CAD CD20 + cells recovered 4 weeks after the first infusion (9 CD20+ cells/μL, 0.02 μg/mL rituximab). Yet, after the next infusion, they remained suppressed for the scheduled period. In the patient with wAIHA, CD20 + cells re-appeared about 4 weeks after the first infusion (41 CD20 + cells/μL, 0.004 μg/mL rituximab), 4 weeks after the third infusion (21 CD20 cells/μL, 0.1 μg/mL rituximab), and at the end of study visit, 2 months after the last infusion (49 CD20 + cell/μL, rituximab not measurable). Section title: Results Educational score: 2.6086385250091553 Domain: clinical Document type: Other Language: en Therefore, the rituximab dose was escalated to 50 mg every 3 months in the next cohort . Section title: Results Educational score: 4.033327579498291 Domain: biomedical Document type: Study Language: en Three patients received 50 mg fixed dose every 3 months , two patients with CAD and one with wAIHA. Rituximab transiently depleted all CD20 + cells in these three patients. However, only in the patient with wAIHA CD20 + cells remained suppressed for the entire study period (345 days = 11 months and 9 days), while CD20 + cells recovered in the other two patients . Section title: Results Educational score: 4.056683540344238 Domain: biomedical Document type: Study Language: en In one patient with CAD, CD20 + cells recovered 3 weeks after the first infusion (150 CD20 + cells/μL, rituximab concentration 0.03 μg/mL), and 1 week after the second infusion (103 CD20 + cells/μL, rituximab concentration not measurable). In the other patient with CAD, CD20 + cells reappeared every 1–3 weeks (1 week after the first infusion: 270 CD20 + cells/μL and rituximab concentration of 0.01 μg/mL, 1 month after the second infusion: 176 CD20 + cells/μL, rituximab concentration 0.004 μg/mL, 1 week after the third infusion 96 CD20 + cells/μL, rituximab concentration 0.01 μg/mL). Therefore, the rituximab dose was escalated to 100 mg every 3 months. Section title: Results Educational score: 3.9160871505737305 Domain: biomedical Document type: Study Language: en One patient with CAD received 100 mg fixed dose every 3 months. Rituximab infusion depleted CD20 + cells completely after the first infusion and showed sustained depletion over the whole study period. Interestingly, through concentrations of rituximab increased over time. CD20 + cells reappeared 4 months after the last infusion (56 CD20 + cells/μL, rituximab concentration 0 μg/mL). Section title: Results Educational score: 4.019142150878906 Domain: biomedical Document type: Study Language: en We estimated terminal elimination half-lives of rituximab using the last two measurable drug concentrations, which is obviously a limitation. However, estimated half-lives ranged from 51 h (approximately 2 days) to 140 h (approximately 6 days), which is much lower than published half-lives of 21 days ( 40 ). Section title: Results Educational score: 3.6814897060394287 Domain: biomedical Document type: Study Language: en There was a close relationship between rituximab plasma concentrations and the number of CD20 + cells: CD20 + cell counts were below the detection limit if rituximab plasma concentrations were above 0.4 μg/mL in all included patients . Section title: Laboratory parameters and emergency treatment Educational score: 4.081881523132324 Domain: clinical Document type: Study Language: en All patients had been pretreated before this trial. Six patients showed signs of ongoing hemolysis with elevated LDH, bilirubin and decreased haptoglobin concentrations. During the study, one patient who received 5 mg/m 2 needed transfusion of packed red blood cells on two occasions (Hb 7.7 g/dL and Hb 7.5 g/dL) , another patient with 5 mg/m 2 rituximab received emergency treatment with sutimlimab ( 41 ) by the treating hematologists because of signs of hemolysis (Hb 6.5 g/dL, reticulocytes 109.5 G/L, LDH 442 U/L, bilirubin 1.84 mg/dL). One patient (50 mg rituximab) needed a transfusion of packed red blood cells at the end of the trial. One patient (100 mg rituximab) developed endocarditis developed endocarditis 228 days after start of rituximab, which resulted in a reduction of hemoglobin to 8.7 g/dL and an increase in bilirubin. Hemolysis stopped completely in two patients after 20 mg of rituximab, as measured by a normalization of haptoglobin values . Section title: Laboratory parameters and emergency treatment Educational score: 1.629054069519043 Domain: biomedical Document type: Study Language: en Further details on the individual patients’ response can be found in Supplementary material . Section title: Safety Educational score: 2.9757611751556396 Domain: clinical Document type: Study Language: en Two serious adverse events occurred during the study. One non-related serious adverse event was reported in a patient, who had a fall, resulting in a laceration bruise. She was hospitalized overnight for observation. One patient suffered from endocarditis shortly after a vigorous dental cleaning procedure performed at a dentist, which was considered as a possibly related serious adverse event and the same patient also had a prolapsed disc during the study. In general, adverse events were evenly distributed and expectable with regards to the included population and the administered treatment. Section title: Discussion Educational score: 4.182130813598633 Domain: biomedical Document type: Study Language: en The aim of this study was to investigate whether very low doses of rituximab suffice to permanently suppress CD20 + cells in patients with AIHA. In total, 10 patients with AIHA completed this study, in whom we observed highly variable responses to the low-dose rituximab regimens. Rituximab infusions fully depleted CD20 + cells transiently in all except one patient across all dosing groups and types of AIHA. However, in all treatment groups there were patients with permanently suppressed CD20 + lymphocytes and patients in whom the anti-CD20 + therapy was ineffective. To optimize recruitment of patients with a rare disease, patients with all types of AIHA were eligible and we did not prespecify to include equal numbers of patients with CAD or warm AIHA. The surplus of CAD patients in this study may be somewhat surprising, but resulted due to chance. Taking the small sample size into account, comparisons of the results between the different subtypes are exploratory, descriptive and should be done only with caution. That said, in our study we did not observe any obvious differences between the different subtypes of AIHA. Successful CD20 + cell suppression was observed in CAD as well as wAIHA (one CAD patient in the group of 5 mg/m 2 , one CAD patient in the group of 20 mg and one wAIHA patient in the group of 50 mg). The same is true for patients with therapeutic failure. A previous study observed a lower clinical response rate to low dose rituximab in patients with CAD than in patients with wAIHA, probably due to its different pathogenesis and a higher load of CD20 + cells in CAD ( 32 ). The small sample size in our study precluded any inferences on clinical effects, especially among the different subtypes of AIHA. However, some CAD patients may have become available for rituximab treatment because they had come off a previous trial ( 42 ) or its subsequent named patient program ( 43 , 44 ). Section title: Discussion Educational score: 4.14890718460083 Domain: biomedical Document type: Study Language: en The initial successful depletion of CD20 + cells by low rituximab doses may be restricted to peripheral blood and early CD20 + cell reconstitution may be a sign of incomplete removal of these cells from various tissue compartments. One may observe a very close relationship between pharmacokinetics and pharmacodynamics in that regard. Estimated half-lives in our study were considerably shorter (approximately 2–6 days) than those observed in studies using authorized doses (approximately 21 days) ( 40 ). This observation is most likely due to ongoing target-mediated drug disposition and incomplete elimination of CD20 + cells from tissues and consistent with our findings from healthy volunteers ( 33 ). Section title: Discussion Educational score: 4.204374313354492 Domain: biomedical Document type: Study Language: en Target-mediated drug disposition refers to the process by which a monoclonal antibody binds to its target, i.e., the CD20 + cells in case of rituximab, with high affinity and builds complexes. These monoclonal-antibody-target complexes are subsequently eliminated by the immune system. Consequently, clearance of the antibody may vary depending on the antigen mass and results in non-linear elimination, especially at low doses ( 18 , 45 ). In patients with malignant diseases, higher rituximab concentrations and longer half-lives are associated with better survival rates, likely because malignant CD20 + cells were completely depleted, which reduces target-mediated drug disposition ( 18 ). In that context, we observed that in patients in whom CD20 + cells recovered after initially successful depletion these recoveries became less pronounced after each consecutive rituximab dose. It is likely that each rituximab dose depletes part of the CD20 + cell pool in tissue and that over time lower doses may suffice to completely remove these cells from all tissues. Section title: Discussion Educational score: 4.140047550201416 Domain: biomedical Document type: Study Language: en Another interesting observation was that rituximab plasma-concentrations of >0.4 μg/mL resulted in a complete depletion of CD20 + cells from peripheral blood in all patients. This plasma concentration may be interpreted as the empirically measured EC95% of rituximab in vivo . These results confirm our initially hypothesized EC95% of >0.6 μg/mL ( 33 ) and support that a trough concentration of >1 μg/mL should be sufficient to permanently deplete CD20 + lymphocytes. Section title: Discussion Educational score: 4.14615535736084 Domain: biomedical Document type: Study Language: en However, we observed a high variability in CD20+ cell suppression in the different patients, which may be attributed to various reasons ( Supplementary discussion ). To overcome the observed variability in CD20 + cell suppression, a rituximab loading dose that depletes the entire CD20 + cell pool reservoir may be necessary. Maintenance doses that serve the purpose of a long-lasting CD20 + cell suppression may be much lower, while being equally effective ( 46 ). The dosing regimen may then be adapted to the individual needs of a patient, i.e., aligned with hospital visits that may differ between various diseases, different patients and circumstances of local healthcare systems. Therapeutic drug monitoring (or monitoring of CD20 + cells) may be useful to ensure effective treatment with the overall aim of plasma concentrations that exceed the EC95% of 0.4 μg/mL or a complete CD20 + cell suppression. Section title: Discussion Educational score: 4.153377056121826 Domain: biomedical Document type: Study Language: en Our sample size was too small to draw definite conclusions on clinical efficacy of low-dose rituximab in AIHA. The main aim of this study was to investigate the pharmacokinetics and pharmacodynamics of various low-dose rituximab regimens focusing on CD20 + cell suppression and the study was not designed to prove therapeutic efficacy. Moreover, our dosing regimens were only partly effective in suppressing CD20 + cells. Nevertheless, previous studies ( 31 , 32 ) have already shown that lower doses than those approved ( 36 , 47 ) do work in AIHA. In a phase II trial ( 31 ) a fixed dose of 100 mg rituximab was administered once a week along with 1 mg/kg/day prednisolone. Response [primary outcome defined by increase in Hb levels and normalization of hemolytic parameters ( 31 )] was seen in all 14 patients with warm AIHA after 2 months and was sustained for 12 months. In the 9 CAD patients an overall response rate of 56% was achieved after 2 months with 11–33% relapse rates at 6 and 12 months. Moreover, steroids could be stopped in 65% of warm AIHA and 56% of CAD patients. A follow up study ( 32 ) further evaluated the sustained response. Relapse free survival in warm AIHA was 90, 100, 100, and 89% at month 6, 12, 24, and 36 respectively, whereas response was slightly lower in CAD (87, 79, 68, and 68% at 6, 12, 24, and 36 months). Section title: Discussion Educational score: 4.041348457336426 Domain: biomedical Document type: Study Language: en The observed rituximab levels in our patient receiving 100 mg rituximab together with the permanent suppression of 3 months in patients with autoimmune hemolytic anemia (AIHA) to effectively su CD20 + cells support Barcellini’s findings ( 31 , 32 ). We extend them by showing that our proposed dosing regimen (100 mg every 3-month) may suffice to maintain a long-lasting CD20 + B-lymphocyte depletion. However, this must be confirmed in larger studies. Section title: Discussion Educational score: 3.9283816814422607 Domain: biomedical Document type: Study Language: en Such a dosing regimen could offer economical advantages, also given that the smallest available vial size is 100 mg. Barcellini’s data ( 31 , 32 ) indicate a highly cost-efficient treatment option for resource limited countries in an off-label condition, where nobody is bound to a specific dose. On a further note, the availability of subcutaneous rituximab formulations may further contribute to improving patient comfort by enabling decentralized, long-term drug administration. However, at the moment, only syringes containing 1,400 mg rituximab are available, which currently limits this option. Section title: Study limitations Educational score: 4.109434127807617 Domain: biomedical Document type: Study Language: en The main limitation of this study is the small sample size. Conclusions should therefore be drawn with caution and further clinical trials to support the data would be desirable. The study focused on the pharmacokinetics and pharmacodynamics of rituximab at very low doses and the study duration for the individual patient was relatively short, which additionally precluded conclusions on clinical efficacy. Half-lives were approximated using only the last two available drug concentrations. Another limitation of all interpretations of CD20 + cell depletion and reconstitution, is the unknown CD20 + cell pool in tissue. We included patients with different forms of AIHA. Differences in the pathophysiology between the subtypes introduce heterogeneity, especially as CAD is associated with clonal B-cell disorders. Also, we only included patients with AIHA. Conclusions for other autoimmune diseases should therefore be drawn with caution. The current data do not provide sufficient evidence to treat patients with low-dose rituximab regimens and future clinical trials are necessary to investigate their clinical efficacy. Section title: Study limitations Educational score: 4.060214042663574 Domain: biomedical Document type: Study Language: en In conclusion, low doses of rituximab transiently depleted CD20 + cells in almost all patients, but the tested low-dose regimens were able to permanently suppress CD20 + cells in only a few patients. Additionally, we empirically identified the in vivo EC95% to be at 0.4 μg/mL, which may guide future studies using low-doses of rituximab. | Review | biomedical | en | 0.999997 |
PMC11695361 | Section title: Introduction Educational score: 4.1869096755981445 Domain: biomedical Document type: Review Language: en Stroke is a clinical syndrome caused by focal or systemic brain damage that lasts more than 24 h or results in death. In highly developed countries, stroke is the third most common cause of adult death, the second most common cause of dementia, and the most common cause of disability ( 1 ). Lifestyle, hypertension, hyperlipidemia, blood glucose, obesity, and obstructive sleep apnea are risk factors for stroke ( 2 ). Stroke is the leading cause of disability worldwide and the second leading cause of death. The Global Stroke Factsheet released in 2022 reveals that lifetime risk of developing a stroke has increased by 50% over the last 17 years and now 1 in 4 people is estimated to have a stroke in their lifetime. In 2021, stroke was the third most common GBD level 3 cause of death (7.3 million [95% UI 6.6–7.8] deaths; 10.7% [9.8–11.3] of all deaths) after ischemic heart disease and COVID-19, and the fourth most common cause of DALYs (160.5 million [147.8–171.6] DALYs; 5.6% [5.0–6.1] of all DALYs) ( 3 ). Section title: Introduction Educational score: 4.020835876464844 Domain: biomedical Document type: Study Language: en There is a large body of evidence that lifestyle factors significantly affect the incidence of stroke and that reducing sedentary time and increasing exercise are necessary changes that can benefit people ( 4 , 5 ). Studies have pointed to a significantly greater prevalence of physical inactivity among stroke survivors ( 6 ). Inflammation is a response triggered by damage to living tissue. Growing evidence for the double-edged role of the immune system in the pathophysiology of stroke ( 7 ). A study points to inflammatory cytokines and cells as potential markers for stroke patients in intensive care units ( 8 ). It has also been suggested that elevated baseline C-reactive protein levels are associated with an increased risk of ischemic stroke ( 9 ). A meta-analysis of individual participant data suggests that blood markers of inflammation are independently associated with vascular recurrence after a stroke ( 10 ). Section title: Introduction Educational score: 3.8831403255462646 Domain: biomedical Document type: Study Language: en Physical activity is a potential modulator of the inflammatory process, which reduces inflammation and thus reduces the incidence of many diseases, such as multiple sclerosis ( 11 ). Other evidence also suggests that increased exercise is associated with reduced inflammation ( 12 , 13 ). Sedentary behavior is associated with increased inflammation in the body. Sedentary behavior causes significant pro-inflammatory effects ( 14 ). Sedentary men with metabolic syndrome have reduced inflammation after an exercise intervention, as evidenced by a reduction in hs-CRP levels ( 15 ). Section title: Introduction Educational score: 3.9803178310394287 Domain: biomedical Document type: Study Language: en This study examined the relationship between physical activity, sedentary behavior and stroke and assessed whether C-reactive protein could act as a mediator to mediate the relationship between physical activity, sedentary behavior and stroke by surveying a study population aged 60 years and older from the National Health and Nutrition Examination Survey (NHANES) over the past 4 years . Section title: Study population Educational score: 3.437138319015503 Domain: biomedical Document type: Study Language: en The data analyzed in this study comes from the National Health and Nutrition Examination Survey (NHANES), a comprehensive population-based survey designed to collect data on the American civilian population. Since 1999, NHANES has collected data on approximately 10,000 individuals in 2-year cycles and has used a multistage probability sampling design to produce a representative sample of Americans in non-institutionalized households. We used data from two cycles in NHANES . Participants with missing stroke information and invalid responses were excluded from data merging, while samples with missing basic information and C-reactive protein were excluded, resulting in 3,010 participants being included in our study . Section title: Assessment of stroke outcome Educational score: 3.7093265056610107 Domain: biomedical Document type: Study Language: en The purpose of this study, we defined stroke outcome as a self-reported positive diagnosis of stroke by a physician. All participants completed a disease-related health status questionnaire before their physical examination. Study participants were asked, “Has a doctor or other health professional ever told you that you had a stroke?” A “yes” answer to this question was coded as stroke-positive, and a “no” answer was coded as stroke-negative ( 16 ). Section title: Measurement of C-reactive protein Educational score: 3.960123300552368 Domain: biomedical Document type: Study Language: en This method quantifies CRP by a latacy-enhanced turbidimetric method. For CRP quantification, a particle consisting of a polystyrene core and a hydrophilic shell was covalently linked to an anti-CRP antibody. Data reduction of the signal was performed by data reduction of the signal on the calibration curve using a storable logit-log function. Quantitative CRP was measured on a Behring nephelometer. The specific process can be found at https://wwwn.cdc.gov/Nchs/Nhanes/2009-2010/CRP_F.htm . Section title: Assessment of sedentary behavior and physical activity Educational score: 3.984950065612793 Domain: biomedical Document type: Study Language: en The Physical Activity Questionnaire (prefix PAQ) is based on the Global Physical Activity Questionnaire (GPAQ) and includes problematic activities related to daily living, leisure time activities and sedentary activities. PA information was collected using a global PA questionnaire based on the one created by the World Health Organization (WHO) ( 17 ). Participants were asked to report their PA behaviors in the past 30 days 1 . Levels of three PA types were examined: strenuous work activity/recreational activity, moderate work activity/recreational activity, and walking/cycling activity. The number of days per week they engaged in each type of PA in a typical week was reported and the amount of time (in minutes) spent on that type of activity during the day. The frequency and duration of these activities were used to calculate weekly metabolic equivalent (MET) estimates. NHANES provides MET corresponding to each activity category to determine activity intensity. Section title: Assessment of sedentary behavior and physical activity Educational score: 4.08163595199585 Domain: biomedical Document type: Study Language: en First, Met/week was calculated by multiplying the total number of minutes per week for each activity by the NHANES-recommended MET value, PA (MET-minutes/week) = MET × weekly frequency × duration of each physical activity ( 18 ), and the sum of all activities was calculated by summing over all activity categories. Next, respondents were categorized according to the US PA guidelines for adherence (moderate-intensity PA for adults should be performed for 150 min per week [equivalent to 600 min/week]). Participants were categorized as physically inactive individuals (600 < MET minutes/week, less than PA recommendations) and physically active individuals (≥600 MET minutes/week, PA recommendations) ( 19 ). The NHANES Sedentary Behavior Assessment is a self-report question. Participants heard questions about how much time in a typical day you usually spend sitting or lying down at work, at home, or at school. This included sitting at a desk, with friends, traveling by car, bus or train, reading, playing cards, watching TV, or using a computer. Self-reported sitting or lying down time is divided into 4 levels (<4 h/d, 4–<6 h/d, 6–8 h/d, and >8 h/d) ( 20 ). Section title: Covariates Educational score: 3.6974825859069824 Domain: biomedical Document type: Study Language: en The following variables were included according to the purpose of the study. Gender (male/female), age (20–39, 40–59, and 60–80), race (Mexican American, other Hispanic, non-Hispanic white, non-Hispanic black, and other race), educational (<high school, high school, and >high school) and marital status (not living alone [Married/Living with Partner]/living alone [Widowed/Divorced/Separated/Never married]). Other confounding variables included smoking (defined as having smoked at least 100 cigarettes in a lifetime) and alcohol consumption (defined as having had at least 12 drinks in a year). Diabetes and hypertension were derived from self-reported physician diagnoses (yes/no). The body mass index is categorized into three categories (<25.0, 25.0-30.0, ≥30.0), and the poverty income ratio is an index of the ratio of household income to poverty. Section title: Statistical analyses Educational score: 4.090731143951416 Domain: biomedical Document type: Study Language: en Given the complex multi-stage (stratified and whole group) sampling design of NHANES, appropriate weights were used. Empowerstats 2 was used for weighted analyses. Sample weights for the Mobile Examination Centre (MEC) interviews were reweighted to combine the total NHANES survey data for the 4 years from 2007 to 2010. For baseline characterization, weighted means (95% CI) were used for continuous variables, observations and percentages (weighted) were used for categorical variables, p -values were calculated using appropriate weights, weighted linear regression was used for continuous variables, and weighted chi-square tests were used for categorical variables. Multivariate logistic regression analyses of the association between physical activity, sedentary behavior and stroke were performed and 95% confidence interval (CI) and odds ratio (OR) were calculated. Variables in the crude models were unadjusted; model 1 was adjusted for gender, age, and race, and model 2 was adjusted for gender, age, race, education, marital status, smoking status, drinking status, hypertension, diabetes, body mass index, and poverty income ratio. Mediation effects analyses were calculated using EmpowerStats (see Footnote 2) software. Mediated effects analyses allow us to calculate mediating effects and are an ideal strategy for elucidating pathways and providing statistical evidence for mechanistic analyses. In this study, direct effects represent associations between sedentary behavior, physical activity and stroke; indirect effects, i.e., associations between sedentary behavior, physical activity and stroke are mediated by inflammatory markers; and mediation ratios represent the percentage of mediated effects. p < 0.05 indicates a significant difference. Section title: Baseline characteristics of the study population Educational score: 3.9448580741882324 Domain: biomedical Document type: Study Language: en Table 1 shows the characteristics of the study population by stroke status. The final sample consisted of 3,010 individuals, of whom 244 (7.53%) had experienced a stroke. The mean age of the included study population was 69.80 years, and the mean age of the stroke population was 73.02 years, with 1,513 (45.67%) males and 1,497 (54.33%) females. In the stroke population, 130 (43.54%) were male. Additionally, p -values calculated based on weighting showed statistically significant differences ( p < 0.05) between age, education, hypertension, diabetes, physical activity, sedentary behavior, poverty income ratio, C-reactive protein, and stroke. There was no statistically significant association between gender, race, marital status, smoking status, drinking status, body mass index, and stroke ( p > 0.05). Section title: Weighted logistic regression results between sedentary behavior, physical activity and stroke Educational score: 4.08671760559082 Domain: biomedical Document type: Study Language: en Table 2 shows the relationship between physical activity and stroke. <600 MET-minutes/week was used as the reference. In the crude model, the OR and 95% CI for stroke in older adults in the ≥600 MET-minutes/week group was 0.486 (0.360, 0.655); after adjusting for gender, age, and ethnicity, the OR and 95% CI for stroke in older adults was 0.548 (0.413, 0.727); when adjusted for all variables included, the results remained statistically significance [0.622 (0.443, 0.875), p = 0.009]. Table 3 shows the relationship between sedentary behavior and 60 years old, using 0 to <4 h/day as a reference. Sedentary behavior was found to be positively associated with the risk of 60-year-olds in the >8 h/day group in the crude model, with an OR and 95% CI of 2.549 (1.517, 4.284); the result was still significant after adjusting for all included variables [2.602 (1.557, 4.348), p = 0.003]. Figure 2 shows the results of smoothed curve fitting for the association of physical activity (A), sedentary behavior (B) and stroke. The smoothing curve shows a decrease in stroke risk as the amount of time spent exercising increases and then enters a plateau period. In contrast, the longer the sedentary time, the higher the risk of stroke, with a continuing upward trend. Section title: Threshold effect analysis Educational score: 4.078135013580322 Domain: biomedical Document type: Study Language: en Table 4 shows the results of the threshold effect analysis of the relationship between physical activity, sedentary behavior and stroke in older adults. The results showed that there was a threshold effect between physical activity, sedentary behavior and stroke, with thresholds of 480 MET minutes/week and 90 min for physical activity and sedentary behavior, respectively, and a significant threshold effect with stroke was found only for physical activity (Log-likelihood ratio < 0.001). Section title: Mediation analysis Educational score: 4.073460578918457 Domain: biomedical Document type: Study Language: en Table 5 shows the weighted mean and 95% CI of C-reactive protein in different physical activity and sedentary behavior in older adults, with higher C-reactive protein levels in low physical activity levels and high sedentary behavior. The mediating role of C-reactive protein in the relationship between physical activity , sedentary behavior and stroke in people aged 60 years and over was explored (sedentary behavior and physical activity were continuous variables). Adjusted for gender, age, race, education, marital status, smoking status, drinking status, hypertension, diabetes, body mass index, and poverty income ratio. A significant mediating effect ( p < 0.05) was found only in sedentary behavior, with a mediation proportion of 3.64%. Section title: Discussion Educational score: 3.891221523284912 Domain: biomedical Document type: Study Language: en We found that the risk of stroke was lower among people aged 60 years and older who met the recommended exercise schedule of the US PA and that sedentary behavior was positively associated with the risk of stroke. C-reactive protein mediated the association between sedentary behavior and stroke in older adults with a mediator ratio of 3.64%. Section title: Discussion Educational score: 4.085176467895508 Domain: biomedical Document type: Study Language: en Most studies have shown that physical activity reduces the risk of stroke. In a standardized case–control study of the global burden of stroke from various risk factors in 22 countries worldwide, the promotion of physical activity was found to significantly reduce the burden of stroke, [odds ratio (OR: 0.69, 0.53–0.90) and population-attributable risk (PAR: 28.5%, 14.5–48.5)]. In a study on stroke, it was noted that acute cerebral ischemia showed a distinct, slight pattern of intra-parenchymal hemorrhage differences in the onset time of weekdays versus weekends/holidays, which may be related to variations in physical activity and stressful times ( 21 ). The existing literature tends to suggest that sedentary behavior is associated with an increased risk of stroke. Sedentary behavior is linked to stroke risk. Among people under 60 years of age with low activity levels, recreational sedentary time over 8 h per day is associated with an increased risk of long-term stroke ( 4 ). A Sedentary lifestyle increases the risk of stroke ( 22 ), prolonged sedentary time is associated with more severe stroke symptoms ( 23 ). Section title: Discussion Educational score: 4.217192649841309 Domain: biomedical Document type: Study Language: en Previous studies have found that inflammation appears to play a crucial role in the survival and recovery after ischemic stroke ( 24 ). Blood markers of inflammation are independently associated with vascular recurrence after stroke in a meta-analysis of individual participant data ( 10 ). In a study of the relationship between pro-inflammatory cytokines and the risk of recurrent stroke, it was demonstrated that inflammatory markers associated with the acute phase response (IL-6, TNF- α , C-reactive protein, and fibrinogen, but not IL-18), were associated with the risk of stroke recurrence ( 25 ). C-reactive protein has been shown to be a prognostic marker after lacunar stroke. In patients with recent lacunar stroke, high sensitivity C-reactive protein (hsCRP) levels predict the risk of recurrent stroke and other vascular events ( 26 ). Inflammation plays an important role in the pathophysiology of stroke, and it has been suggested that inflammatory risk markers within neutrophils, lymphocytes and C-reactive protein may have a strong independent predictive value for stroke outcome ( 27 ). The mediating effect of CRP explained only 3.64% of the relationship between sedentary behavior and stroke, which still suggests that sedentary behavior may affect stroke risk partly through inflammatory pathways. This finding suggests that sedentary behavior, even though a small inflammatory response, can still have a potentially negative impact on health. Therefore, it may serve as a basis for early identification and intervention in clinical settings. Section title: Discussion Educational score: 4.119075775146484 Domain: biomedical Document type: Study Language: en Sedentary behavior is strongly associated with elevated levels of cytokines, which are also involved in many regulatory and inflammatory processes ( 28 , 29 ). Previous studies have revealed the importance of cytokines in immune regulation, tissue repair and inflammatory diseases. In particular, the overexpression of some cytokines has been implicated in the pathogenesis of stroke ( 30 ), which makes exploring the relationship between sedentary behavior and cytokines an important direction in exploring the mechanisms of stroke genesis ( 31 ). This implies that sedentary behavior may influence the risk of stroke by affecting the degree of inflammatory response ( 32 ). The results of the mediation analyses suggest that C-reactive proteins play a significant regulatory role in the relationship between sedentary behavior and stroke. This finding triggered our interest in more in-depth studies on the role of inflammatory biomarkers in stroke pathogenesis. Further studies could explore the interactions between different inflammatory markers and how they play a role in sedentary behavior affecting stroke risk. This deeper dug information will help to understand the relationship between lifestyle and stroke more comprehensively and provide a more scientific basis for the development of prevention and intervention strategies. Section title: Discussion Educational score: 3.9652013778686523 Domain: biomedical Document type: Review Language: en Reducing sedentary behavior and increasing physical activity are effective ways to prevent stroke in older adults. A study of physical activity and stroke risk in middle-aged and older adults found negative associations between intensity, frequency, duration, and volume of physical activity and stroke risk in middle-aged and older adults ( 33 ). Physical activity is recognized as an effective intervention to improve psychosocial well-being after stroke ( 34 ). Exercise intervention programs improve frailty and cognitive traits, thereby optimizing functional capacity during the aging process, sedentary lifestyles are associated with declining muscle function and cardiorespiratory fitness, leading to impaired ability to perform daily activities and maintain independent functioning, and exercise is an alternative to medication ( 35 ). Section title: Discussion Educational score: 4.078306198120117 Domain: biomedical Document type: Study Language: en Our study has several strengths. First, we used a nationally representative NHANES cohort, adjusted for confounders (including socio-demographic and lifestyle factors), and considered the complex sampling of NHANES in the analytical calculations. Secondly, the possible mediating role of inflammatory markers was considered when exploring the relationship between sedentary behavior, leisure physical activity and stroke, and C-reactive protein was found to significantly mediate the relationship between sedentary behavior and stroke in older adults. Finally, we also recognized that reducing sedentary time and increasing physical activity appeared to alter inflammatory status, and therefore, understanding the extent and specificity of the relationship between blood cell-based inflammatory biomarkers and stroke has further health implications. Section title: Discussion Educational score: 3.7215073108673096 Domain: biomedical Document type: Study Language: en There are several limitations to the study in this article. First, the data on physical activity and stroke came from a questionnaire, and the quantification of activity time by recall is obviously subjective, and more rigorous measurements, such as wearable physical activity monitor (PAM)-based physical activity measurements ( 36 ), should be adopted in subsequent studies; in addition, the physical activity equivalents calculated in this study were based on overall activity levels, and future studies could explore the effects of different types of physical activity levels on stroke ( 16 ); and finally, the source of this data is the United States, and caution should be exercised when extrapolating to the total population. Section title: Conclusion Educational score: 3.2188990116119385 Domain: biomedical Document type: Study Language: en In people aged 60 years and older, sedentary behavior was positively associated with stroke, whereas physical activity was negatively associated with stroke, and C-reactive protein mediated the relationship between sedentary behavior and stroke. | Review | biomedical | en | 0.999997 |
PMC11695362 | Section title: Introduction Educational score: 4.3698296546936035 Domain: biomedical Document type: Review Language: en Breast cancer, that originates from the epithelial cells of the breast ducts and lobules, is the most common cancer affecting women. It is estimated that women in developed countries have about 1 in 8 chance of developing breast cancer in their lifetime ( 1 ). Triple-negative breast cancer (TNBC) is an aggressive and metastatic form that accounts for 15–20% of all breast cancers ( 2 ). TNBC cells lack estrogen receptor (ER), progesterone receptor (PR), and the human epidermal growth factor receptor 2 (HER2), making them very difficult to treat and contributing to their poor prognosis ( 3 ). Current breast cancer treatments include surgery, chemotherapy, and radiotherapy but these methods are often associated with adverse effects such as cosmetic damage, pancytopenia, nausea, diarrhea among others ( 4 ). Over the years, immunotherapy has gained approval for breast cancer treatment, with vaccines and immune checkpoint blockade being employed ( 5 ). Thus far, adoptive cell therapy, including chimeric antigen receptor (CAR) T cell immunotherapy has shown clinical promise in hematological malignancies such as leukemia, lymphoma, and multiple myeloma ( 6 ). Section title: Introduction Educational score: 3.9757964611053467 Domain: biomedical Document type: Review Language: en CAR-T cell immunotherapy involves engineering a patient’s T cells to express a chimeric antibody-T cell receptor, thereby redirecting them for effective tumor targeting without the need for MHC ( 7 ). However, the clinical efficacy of CAR-T cells for most solid tumors is substantially limited due to insufficient trafficking, poor functional persistence and inhibition by the immunosuppressive tumor microenvironment ( 8 ). To address these challenges, CAR-T cell therapy has been combined with other clinically approved treatments, including radiotherapy ( 9 ). Section title: Introduction Educational score: 4.328597545623779 Domain: biomedical Document type: Review Language: en Image-guided radiation therapy (IGRT) is an approved therapeutic procedure used in combination regimens for managing various malignancies ( 10 , 11 ). This procedure alters the tumor microenvironment (TME) by disrupting its mechanical and functional barriers, leading to the release of proinflammatory cytokines that can activate systemic immunomodulatory effects beneficial for CAR-T cells ( 12 ). Recent immunotherapy strategies have combined radiation with immune checkpoint inhibitors to enhance immune responses against solid tumors ( 13 , 14 ). Targeted radiotherapy also causes the release of death-associated molecular patterns (DAMPs), that stimulate immune system activation, including vascular remodeling, neoantigen expression, and endothelial cellular adhesion changes that promote immune cell infiltration into the tumor ( 15 ). Although CAR-T cell and IGRT therapies have limitations as single-agent treatments for solid tumors, IGRT targets the TME of imageable tumors but misses micromets while CAR-T cells can reach both primary tumors, and micrometastases but are often ineffective due to immunosuppressive TME ( 16 , 17 ). Hence their combination can create a synergistic effect that enhances therapeutic efficacy. Section title: Introduction Educational score: 4.235201358795166 Domain: biomedical Document type: Study Language: en Carcinoembryonic antigen (CEA or CEACAM5) is an oncodevelopmental cell surface glycoprotein identified with the Cluster of Differentiation designation CD66e ( 18 ). It is a tumor-associated protein that is highly expressed in various solid tumors, including colon, gastrointestinal, breast, and lungs ( 19 ). CEA plays a significant role in tumor detection, prognosis, treatment monitoring, and its upregulation associated with the progression, proliferation and migration of metastatic breast tumors ( 20 ). Since most anti-CEA antibodies cross-react with other members of the CEA family found in normal tissues, the use of a CEA specific antibody such as our humanized M5A antibody is critical ( 21 ). Our previous study in immunocompetent CEA transgenic (CEA-Tg) mice that express the same antigen as human in endogenous organs demonstrated specific CAR-T cell responses in CEA positive breast and colon tumor models without any observed off-target effects ( 22 ). Similarly, Chmielewski and colleagues used anti-CEA CAR-T therapy in CEA-Tg mice bearing pancreatic tumors with no evidence of destruction of CEA-positive normal tissues ( 23 ). Section title: Introduction Educational score: 4.117115020751953 Domain: biomedical Document type: Study Language: en Currently, most CAR-T constructs utilize a single-chain variable fragment (scFv) antigen-binding domain that have a tendency to aggregate and reduce the antigen-antibody interaction ( 24 ). This aggregation can result in the formation of CAR aggregates on the surface of CAR-T cells, leading to unexpected signaling and constitutive activation of T cells through an antigen-independent mechanism known as tonic signaling. This phenomenon contributes to faster signal loss and reduced efficacy of CAR-T therapy ( 25 ). Alternatives to scFv-based CARs, such as Fab-based chimeric antigen receptor T targeting CD276 ( 26 ) and CD19 ( 27 , 28 ), have been explored and tested in vitro as substitutes for scFv CAR-T cells. Section title: Introduction Educational score: 4.115175247192383 Domain: biomedical Document type: Study Language: en In this study, we assessed the specificity and functional killing efficacy of scFab anti-CEA CAR-T cells against CEA-positive TNBC cells both in vitro and in a xenograft metastatic tumor model. We hypothesized that antigen derived expansion of CAR Ts would occur at both the primary and metastatic sites and benefit from IGRT directed only at the primary site. Section title: Generation of CAR-specific T cells Educational score: 4.237596035003662 Domain: biomedical Document type: Study Language: en The M5A-targeted 28z CAR construct consists of an hM5A(Fab) domain linked to an IgG4 hinge region with CD28 transmembrane and co-stimulatory domains, and the intracellular signaling domain of CD3ζ . In contrast, the 4-1BBz CAR construct includes an hM5A(Fab)-IgG4-derived Fab linked to the transmembrane domain of CD4, the intracellular domain of 4-1BBz, and the CD3ζ intracellular signaling domain . The two scFab CAR-T plasmids, driven by the EF1p promoter, feature distinct signaling domains; one with a CD28-CD3zeta configuration and the other with a 4-1BB-CD3zeta configuration. Both constructs include a T2A ribosomal skip sequence that separates the codon-optimized CAR sequence from the truncated human CD19t (hCD19t) to allow for identification and enrichment of expressed CARs. . Leukapheresis products were obtained from healthy donors under COH-approved protocols. Peripheral blood mononuclear cells (PBMCs) were separated using density gradient centrifugation on Ficoll-Paque and depleted of CD14+ and CD25+ cells. T naïve/memory cells were then selected from the resulting negative fraction using CD62L+ magnetic beads and activated with CD3/CD28 beads. Activated cells were transduced with the different CAR lentiviral vectors and expanded as previously described ( 22 ). Similarly, the lentiviral plasmid construct used for dual transduction of luciferase CAR-T cells was generated using the epHIV7 vector, with luciferase expression driven by the EF1α promoter. Section title: Antibodies and flow cytometry Educational score: 4.099392890930176 Domain: biomedical Document type: Study Language: en Flow cytometry was performed for the phenotypic analysis of T cells and/or tumors. Briefly, CAR-T cells (1 × 10 5 ) were suspended in FACS staining solution and incubated with fluorescently labeled antibodies for 15 min at 4°C. Unless otherwise stated, cells were stained with mouse anti-human CD3, CD8, CD4, PD-1, Tim3 and CD19 antibodies (BD Biosciences). Anti-CD45RA and anti-CD62L antibodies (BD Biosciences) were used to assess the differentiation status of CAR-T cells. Section title: Effector: target cell killing assay Educational score: 4.1489739418029785 Domain: biomedical Document type: Study Language: en Mock or anti-CEA CAR-T cells were incubated with either WT-MDA-MB231 (ATCC; HTB-26) or MDA-MB231 transfected with CEA and GFP ( 29 ) at varying effector to target (E:T) ratios (0.5:1, 1:1, 2:1, 4:1). Briefly, 100 µl of target cells in fluoroBrite DMEM complete medium was first seeded in 96-well plates, followed by addition of 100 µl of effector cells to the same well. The mixture was incubated overnight at 37°C for 24 hrs. Target-only cells were used as controls. The percentage cell cytotoxicity was calculated following the formula shown below, where 100% cell viability (value max ) was measured by averaging the fluorescence readings of the target cells without any T cells. The fluorescence for each well co-cultured (target and effector) is labeled as the experimental value (value exp ). The background was subtracted as (value min ) from both value max and value exp . The fraction of live cell fluorescence was calculated by dividing ( v a l u e exp − v a l u e min ) by ( v a l u e max − v a l u e min ). To determine the fluorescence loss due to GFP-expressing target cell death, this fraction was subtracted from 1. The resulting value was then multiplied by 100% to obtain the percentage of cell cytotoxicity. Section title: Cytokine measurement Educational score: 4.092537879943848 Domain: biomedical Document type: Study Language: en Cytokine release was analyzed using an ELISA kit assay according to the manufacturer’s instructions. In brief, 50 µL of supernatant from the co-culture killing assay was collected and diluted 1:10 with the kit buffer solution. Cytokine levels for IFN-γ and granzyme B were measured using a human ELISA kit, with absorption readings in triplicates taken on a CLARIOstar instrument. Section title: Measurement of cellular degranulation and T-cell exhaustion Educational score: 4.131464004516602 Domain: biomedical Document type: Study Language: en CD107a was used to assess the level of cellular degranulation, a prerequisite for T-cell mediated cytolysis. Briefly, target cells were co-cultured with anti-CEA CAR-T cells for 6 hours at an effector-to-target (E:T) ratio of 2:1, with 0.26% w/w of Golgi stop added to the media. After the incubation period, the CAR-T cells were collected and stained with 1:20 dilutions of anti-CD3, CD4, CD8, and CD107a antibodies for 25 minutes on ice. The cells were then washed and analyzed using a flow cytometer. Section title: Measurement of cellular degranulation and T-cell exhaustion Educational score: 4.060637474060059 Domain: biomedical Document type: Study Language: en To measure T cell exhaustion, target cells and scFab CAR-T cells were co-cultured at an effector-to-target (E:T) ratio 2:1 for three days. After the incubation period, the cells were washed with 1% PBS and subsequently stained with PD-1 and TIM-3 markers. Section title: Animal study Educational score: 4.132370948791504 Domain: biomedical Document type: Study Language: en All animal studies were performed with NOD/SCID/IL-2rg . Animals were housed in pie cages, in a pathogen free room with a maximum of 5 mice per cage. On Day 1, mice were engrafted orthotopically with 5x10 5 MB231/CEA-Luciferase positive cancer cells in 50 μL of PBS and Matrigel solution at 1:1 ratio into the mammary fat pat of the female mice using 28G Insulin Syringes . MB231/CEA-Luciferase positive cells were used to monitor tumor metastases using anti-firefly luciferase. Tumor size was measured and established tumors (50-75 mm 3 ) on day 9 were randomly assigned to groups (n=4-5 mice per group). On day 10 post tumor engraftment, both radiations only group and combination treatment groups received 10 Gy of irradiation each. On day 11, combination group and CAR-T groups only were treated with 1x10 6 scFab anti-CEA CAR-T cells in 200 μL PBS were injected intravenously. Untreated groups received PBS. Tumor growths were monitored and measured with caliper and growth endpoint were set at >1500 mm 3 . Section title: Animal study Educational score: 4.133438587188721 Domain: biomedical Document type: Study Language: en To track CAR-T cell tumor infiltration, activated human T-cells were co-transduced with lentivirus encoding GFP-firefly luciferase and scFab CAR-T. The double-positive cells were then isolated using a flow cytometry cell sorter. For the experiment, NSG mice bearing MDA-MB-231/CEA tumors (which do not express luciferase) were utilized and followed the same experimental procedure described above. On day 6, the mice were divided into treatment groups: untreated controls, CAR-T cells alone, fractionated radiation (4x 2.5 Gy), or single-dose IGRT (10 Gy). The untreated control group included 2 mice, while the other groups consisted of 3-5 mice each. The fractionated radiation group received 2.5 Gy daily from day 7 to day 10, while the single-dose group was treated with 10 Gy once on day 10. On day 11, both the fractionated and single-dose groups received an intravenous injection of 1x10 6 anti-scFab-CEA CAR-T cells expressing luciferase in 200 μL PBS. Untreated mice served as controls. Mice were imaged weekly using the LAGO bioluminescence imaging system (Spectral Instruments Imaging, LLC, Tucson, USA), and luciferase signals were quantified for comparative analysis. Section title: Tissue collection and analysis Educational score: 4.156712532043457 Domain: biomedical Document type: Study Language: en At endpoint, lungs tissue and tumors were collected in cold PBS. For flow cytometry analysis, small fractions of tumor were cut into pieces and digested with Tumor Dissociation Kit, Mouse (MACS, 130-096-730) and gentleMACS C Tubes (MACS, 130-096-334), as per manufacturer’s directions. The cells were meshed on a 0.40 μM cell strainer and lysed with Red Blood Cell Lysis Buffer Hybri-Max . Thereafter, the cells were stained with fluorescent antibodies for flow cytometry. To measure IFN-γ production, cells were re-stimulated with Cell Activation Cocktail in 10% FBS RPMI media for 4 hours at 37°C. Following, cells were stained for surface markers (CD4, CD8, PD-1) and viability marker (Zombie UV, BioLegend). These cells were fixed and permeabilized with Fixation/Permeabilization kit (ThermoFisher) according to the manufacture’s protocol and stained for intracellular IFN-γ (BioLegend) and analyzed by flow cytometry. Section title: Tissue collection and analysis Educational score: 4.092494964599609 Domain: biomedical Document type: Study Language: en For immunohistochemistry (IHC), the harvested tumor and lungs tissues were fixed with 4% paraformaldehyde for 3 days and thereafter stored in 70% ethanol. Tissues were washed in PBS and frozen on dry ice using O.C.T. in vinyl Specimen molds for H&E and IHC staining. Tissue block slides were stained with anti-firefly (luciferase detection) and mouse anti-human CD3 antibodies. Section title: Statistical analysis Educational score: 3.4657504558563232 Domain: biomedical Document type: Study Language: en The results were analyzed using Prism statistical software using the T-test, one way ANOVA, or two-way ANOVA to compare the two experimental groups. Statistical analysis of more than three groups will be based on two-way ANOVA and Sidak’s multiple comparison test. A threshold of p < 0.05 was considered statistically significant throughout. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Section title: Production of scFab-CAR T cells Educational score: 4.226929664611816 Domain: biomedical Document type: Study Language: en Our initial studies used an all murine anti-CEA scFv CAR T derived from the anti-CEA monoclonal antibody T84.66 in immunocompetent CEA Tg mice ( 22 ). However, a scFv CAR-T derived from the humanized version of this antibody ( 21 ) exhibited poor stability in T cells (data not shown). Consequently, we designed and tested anti-CEA scFab CAR-T cells, that included constant and variable domains from the heavy and light chains of the humanized anti-CEA antibody (M5A) plus signaling domains CD28zeta or 4-1BBzeta . The construct also included an expression cassette for a truncated human CD19 gene (hCD19t) to allowing enrichment of transfected from non-transfected T cells ( 30 ). CD3+ T naïve/memory cells from human donors were transduced with or without (mock – untransduced) lentivirus encoding the anti-CEA scFab, and transduction efficiency was confirmed by flow cytometry monitoring hCD19t expression following enrichment with anti-CD19 beads . CAR-T cells produced from three different human donors (HD) were enriched using anti-CD19 magnetic beads that resulted in final transduction efficiencies of 94%, 89% and 81% for CD28z-CAR-T and 82%, 75%, and 83% for 4-1BBz-CAR-T cells, respectively . The mock transfected T cells showed no CAR-T cell expression. The growth expansion curve of T cells post-CAR-T production from three different donors is shown , demonstrating good activation and proliferation of CAR-T cells. These results indicate that using the scFab fragment of the humanized M5A antibody for CAR-T production does not negatively impact the activity of the transduced human T cells. Section title: Target specificity of scFab anti-CEA CAR-T cells Educational score: 4.361637115478516 Domain: biomedical Document type: Study Language: en To determine the antigen specificity of anti-CEA scFab CAR-T cells, CD28z-CAR-T, 4-1BBz-CAR-T, or mock T cells were incubated with triple-negative human breast cancer cells (MB231) transfected with CEA and GFP or GFP only . These cells were used in increasing Effector: Target (E:T) ratios with CEA or GFP-only cells as positive controls for specific and nonspecific targeting, respectively. The specific lysis of CEA + versus CEA - target cells by scFab CAR-T cells was demonstrated, with the highest killing observed in CD28z-CAR-T cells. In contrast, the mock T cells were ineffective in killing the targets . Measurement of IFN-γ and granzyme B by ELISA in the supernatants from the co-culture killing assay with CEA + target cells showed an increased release of IFN-γ at 4:1 E:T ratio for CD28z-CAR-T cells . Conversely, 4-1BBz-CAR-T exhibited increased release of granzyme B . For control target cells, no IFN-γ or granzyme B was detected. In a separate experiment, the co-culture killing assay was extended for three days, and the expression levels of PD-1 and TIM-3 exhaustion markers were measured. The CD4 and CD8 T cell subpopulations in both CEA - and CEA + MDA-MB231 cells co-cultures showed increased expression of PD-1 but not Tim3 exhaustion markers on the effector cells . The expression of the PD-1 marker was higher in CD28z-CAR-T cells compared to 4-1BBz-CAR-T cells. Furthermore, the expression of CD107a degranulation marker was higher in scFab CD28z-CAR-T cells than in 41BBz-CAR-T cells . Together, these data confirm that both formats of scFab anti-CEA CAR-T cells specifically targeted CEA-expressing cells through antigen recognition and T cell activation. Section title: Image guided radiation therapy enhances the effectiveness of anti-CEA scFab CD28z-CAR-T cell therapy Educational score: 4.223911762237549 Domain: biomedical Document type: Study Language: en To evaluate the therapeutic efficacy of anti-CEA scFab CAR-T cells against solid tumors, MB231/CEA breast tumors were orthotopically implanted in the mammary fat pads of immunocompromised NSG mice. MDA-MB231 xenografts were chosen due to their propensity to spontaneously form lung metastases ( 31 ). Established tumors in mice were treated with a 10 Gy single dose of IGRT alone, scFab CAR-T alone, or IGRT followed by scFab CAR T the following day . A significant tumor growth reduction was observed in the mice treated with the combination of IGRT and anti-CEA scFab CAR-T cells compared to either monotherapy. The combination therapy was statistically significant (p<0.01) compared to scFab CD28z-CAR-T cells alone and untreated control . The 10 Gy IGRT-treated mice showed significantly reduced tumor growth until day 30, after which the tumor growth escaped. On day 48, when the tumors in the control and scFab CAR-T cell-only treated mice reached maximum volume, the tumors and lungs from all mice were collected. Tumor weight measurements post-euthanasia showed a significant difference between the control and combined treatment groups . At this endpoint, portions of the tumors were enzymatically digested and analyzed by flow cytometry for T cell phenotyping. Flow cytometry analysis revealed the presence of anti-CEA CAR-T cells in combination group only, and an increased CD4 T cell subpopulation compared to CD8 . To assess T cell functions such as IFN-γ production and Treg activity, harvested T cells were restimulated for 4 hrs, followed by intracellular staining for IFN-γ and FoxP3. The analysis showed fewer exhausted cells stained with CTLA-4 in CD4 and PD-1 in the CD8 cell population. Additionally, there was an increased number of IFN-γ producing CD8 cells, that likely contributed to the improved anti-tumor activity and tumor inhibition . The expression of PD-1 on CD4+ CAR T cells was only 20% with less than 1% of IFN-γ (data not shown). Repeating the experiment with a different donor T-cell, including mock and 4-1BB variants of the anti-CEA scFab CAR-T cells, also showed similar results with the best tumor inhibition observed in CD28-scFab-CAR-T . Section title: IGRT improves the infiltration scFab anti-CEA CAR-T cells in solid tumors Educational score: 4.146834850311279 Domain: biomedical Document type: Study Language: en Anti-CD3 immunohistochemistry (IHC) analysis of the tumor tissues revealed poor infiltration of anti-CEA scFab CAR-T cells in the CAR-T only treated group compared to their significant infiltration in the combined therapy group . CD3+ cells were abundant in the combination group even 37 days post a single injection of scFab anti-CEA CAR-T cells. Quantification of the infiltrating scFab CAR-T cells in the tumor showed a significant increase in the combination treatment group . These findings suggest that IGRT facilitated the infiltration, persistence, and antitumor response of scFab CAR-T cells within solid tumor tissues. Section title: Combination IGRT with anti-CEA scFab CAR-T therapy prevents breast cancer metastasis to the lungs Educational score: 4.135538101196289 Domain: biomedical Document type: Study Language: en Triple-negative breast cancer is known to metastasize to the lungs, so we performed IHC analysis of lung tissue to detect luciferase-positive tumor cells. Massive metastases were found in the control group, while slightly fewer metastases were detected in the mice treated with 10 Gy IGRT alone. Interestingly, relatively few metastases were observed in the CAR-T only treated mice, while the combination of IGRT and scFab anti-CEA CAR-T therapy almost eliminated lung metastases . Luciferase staining of the lung lobes indicates metastatic lesions, and their quantification showed a significant bigger tumor aera in the lungs in the control group compared to the combination treatment group . A similar pattern was observed when the experiment was repeated with a different CAR-T cell donor and included the scFab 4-1BBzeta variant . These data demonstrate that the combination of IGRT and anti-CEA CAR-T therapy elicits a strong antitumor response against both the primary tumor and distant metastatic spread to the lungs. Section title: Kinetic tracking CAR-T cells tumor infiltration in vivo Educational score: 4.307359218597412 Domain: biomedical Document type: Study Language: en Monitoring CAR-T cell tumor infiltration and proliferation in vivo provides an important insight into kinetics and effectiveness of the therapy. In this study, we co-transduced T cells with two lentiviral vectors expressing anti-CEA scFab CAR and GFP-Luciferase, respectively, to examine the kinetics of CAR T infiltration, preceded by 4 x daily 2.5 Gy IGRT to extend the TME effects over a longer period, as previous described ( 29 ). The treatment schedule is outlined in and production of double-positive (CD19 and GFP-Luciferase) CAR T-cells is shown in . One day following IGRT, the mice were intravenously injected with 1x10 6 anti-CEA scFab CAR-T cells expressing luciferase. CAR-T cells were tracked in NSG mice bearing orthotopic MDA-MB231/CEA tumors (without luciferase) on days 18, 25 and 32 after treatment using bioluminescence. The CAR-T cell bioluminescence was low for all groups at day 18, peaked on day 25, and returned to low values by day 32 that corresponded with objective eradication of the tumors in both combination groups . Interestingly, the bioluminescence data of day 18 showed significantly higher signal in fractionation IGRT+CAR-T group suggesting more efficient TME remodeling. The results for the CAR T only group have consistently low luminescent values demonstrating that few CAR T cells enter the tumor, take a week to expand to their maximum luminescent signal, and thereafter decline. Similar kinetic profiles are seen for the combination therapy groups with highest overall values for the combination therapy. We conclude (a) that the number of CAR T cells arriving at the tumor determine the degree of expansion, explaining the similar kinetics for all three groups, and (b) that IGRT increases that number, explaining the magnitude of the expansion in terms of luminescent signal. These findings underscore the importance of pretreatment of the tumor with IGRT and appropriate timing of the subsequent CAR T therapy. Section title: Discussion Educational score: 3.9090418815612793 Domain: biomedical Document type: Study Language: en CAR-T adoptive immunotherapy has been one of the most successful cellular therapies to date, particularly for hematological malignancies. However, the immunosuppressive TME of solid tumors presents challenges that require strategies to enhance the homing and expansion of CAR-T cells within the TME. Among the approaches to improve CAR-T cell infiltration into solid tumors, low dose radiotherapy, especially low dose image guide radiotherapy stands out ( 32 , 33 ). Section title: Discussion Educational score: 4.137456893920898 Domain: biomedical Document type: Study Language: en Selecting the best tumor antigen for T cell mediated immunotherapy is critical both for efficacy and reducing off target toxicity. CEA was chosen as a tumor antigen due to its broad application in tumor detection, diagnosis, prognosis, and treatment monitoring, and its association with metastatic breast tumors ( 20 ). The humanized anti-CEA antibody M5A has shown efficacy with no toxicity in several clinical trials to date ( 19 , 34 ). A preclinical study with anti-CEA CAR-T cells from another group targeting the A3B3 domain of CEA, also showed specific targeting with no off-target toxicity in CEA transgenic mice ( 23 ). Our in vitro results with our anti-CEA scFab CAR-T cells exhibited good transduction efficiency, activation, and proliferation . The cellular cytotoxicity of scFab CAR against triple-negative breast cancer cells transfected with human CEA (MB231/CEA) vs CEA negative cells showed a high specificity and excellent E:T ratios. Section title: Discussion Educational score: 4.152990818023682 Domain: biomedical Document type: Study Language: en In our initial study using syngeneic mouse anti-CEA CAR-T cells, we used a scFv construct that showed significant activity when combined with IL-2 antibody fusion immunocytokine ( 22 ). Despite its efficacy against a syngeneic solid tumor mouse model, the humanized anti-CEA scFv construct proved unstable in xenograft CAR-T therapy. Consequently, we designed and tested a scFab CAR-T cell, which includes one constant and one variable domain from the heavy and light chain of the humanized anti-CEA antibody M5A ( 21 ). The concept of using scFab fragments stems from numerous reports examining the structure and therapeutic advantages of antigen-specific fragments of antibodies produced through recombinant processes ( 35 ). scFab-CAR-T cells were first tested by Duan and colleagues, who reported that novel Fab-CAR-T cells demonstrated heightened recognition of tumor antigens in human thyroid cancer cells and extended the lifespan of CAR-engineered T cells, generating a durable antitumor response ( 26 ). Section title: Discussion Educational score: 4.104982852935791 Domain: biomedical Document type: Study Language: en In our studies we tested scFab CAR-T in two variants of costimulatory signaling domains CD28 or 4-1BB. Previous studies showed that CD28-based CAR-T cells usually resulted in a more robust proliferative response and effector memory T cells, whereas 4-1BB co-stimulation induced a progressive response and with enhanced persistence and central memory differentiation ( 36 ). Moreover, the work by Starr et al. ( 37 ) showed improved specificity, persistence, and efficacy of 4-1BB–based IL13-ligand CARs when compared to CD28 format ( 37 ). Nonetheless, the selection of the co-stimulatory domain remains controversial and may be influenced by the structure of CAR molecules and the histopathology of the target diseases. However, our in vitro and in vivo studies have proven similar anti-tumor effects for both scFab CAR-T formats. Section title: Discussion Educational score: 4.195350646972656 Domain: biomedical Document type: Study Language: en As expected, treatment of orthotopic TNBC cancer-bearing mice with our anti-CEA scFab CAR-T cells alone did not result in significant infiltration of these cells into the tumor or tumor inhibition. However, combination treatment with IGRT (single low dose of 10 Gy) resulted in synergistic activity, leading to stronger tumor inhibition compared to individual treatments. A repeat of the experiment with a different donor T-cell also showed similar tumor growth inhibition. For CAR-T cells to effectively kill tumor cells, they must sufficiently infiltrate the hostile tumor microenvironment ( 38 ). The combination of anti-CEA scFab CAR-T cells with IGRT increased CAR-T cell infiltration into the tumor as shown by immunostaining and luciferase labeling of the CAR T cells. A similar observation was also reported by Quach and colleagues, who found that tumor-targeted radiation prior to systemic administration of CAR-T cells substantially improved CAR-T cell therapy efficacy and infiltration in solid tumors ( 39 ). Akhavan et al. ( 40 ) investigated the effects of stereotactic radiation therapy at doses of 5, 10, and 20 Gy on the TME in a GBM murine tumor model. They found that a conditioning dose of 10 Gy was particularly effective in stimulating cells to enhance tumor growth kinetics and induce gene expression changes that support the combination with CAR-T cell immunotherapy ( 40 ). Additionally, other groups have reported that administering a sub-cytotoxic radiotherapy dose of 0.5 – 2Gy followed by CAR-T cells infusion have increased the regulation of death receptor molecules to enhance CAR-T cell efficacy ( 41 ). Section title: Discussion Educational score: 4.18089485168457 Domain: biomedical Document type: Study Language: en An alternative approach by Cao and colleagues found that combining microwave ablation radiation therapy with AXL-CAR-T cells resulted in superior antitumor efficacy. Their findings suggest that tumor guided radiation enhances the activation, infiltration, persistence, and tumor-suppressive properties of AXL-CAR-T cells in non-small cell lung cancer patient-derived xenograft tumors via tumor microenvironment (TME) remodeling ( 42 ). Treatment of antigen-heterogenous pancreatic cancer with low-dose radiation therapy and CAR-T cells demonstrated that localized radiation can sensitize antigen-negative tumor cells, which would otherwise evade CAR recognition, to be effectively eliminated by CAR-T cell killing ( 43 ). IGRT prior to immunotherapy can cause tumor TME remodeling and depletion of some immunosuppressive cells, enhancing CAR-T cell migration to the tumor site. Thus, the combination therapy induced significant tumor suppression without observed toxicity in humanized immunocompetent mice. Section title: Discussion Educational score: 4.265072345733643 Domain: biomedical Document type: Study Language: en However, the risk of induction of metastatic spread to distant organs caused by primary tumor irradiation has been less studied. Bouchard et al. ( 44 ) investigated the impact of radiation on the mammary glands, focusing on the invasiveness of breast cancer cells that survive radiation treatment. Their findings revealed a significant increase in breast tumor cell migration from the primary tumor compared to non-irradiated controls. This was associated with elevated expression of pro-migratory and pro-inflammatory molecules such as IL-6, cyclooxygenase-2, membrane type 1 metalloprotease, phospholipase A2, and transforming growth factor-β (TGF-β), which likely facilitated the migration of cancer cells, increased circulating tumor cells, and metastasis to the lungs. Supporting this, Biswas et al. ( 45 ) showed that increased secretion of TGF-β by stromal cells post-irradiation promoted lung metastases in an orthotopic mammary tumor model. Similarly, irradiation of hepatoma cells has been linked to the secretion of tumor necrosis factor-alpha (TNF-α), IL-6, VEGF, epidermal growth factor (EGF), MMP2, and MMP9, all of which enhance tumor invasion ( 46 ). Additionally, radiation has been shown to favor cancer cell migration at the expense of primary tumor growth in a glioblastoma rat model. Brain irradiation before primary tumor implantation promoted the infiltration of cancer cells into distant organs and induced a phenotypic shift in glioma cells from a proliferative to an invasive type ( 47 ). Section title: Discussion Educational score: 4.161683082580566 Domain: biomedical Document type: Study Language: en The impact of vascular changes following IGRT is controversial. For example, Castel and Kirsch ( 48 ) reported that high-dose radiation causes endothelial cell proliferative defects, leading to increased vascular permeability and subsequent tumor cell death. However, Budach and colleagues ( 49 ) found no difference in local tumor control across various human cell lines using the high radiation dose necessary to cure 50% of tumors implanted in nude or SCID mice. Their findings suggest that stromal endothelial cells do not significantly influence tumor control by radiation, despite differences in the radiosensitivity of the mice used, supporting the theory of direct tumor killing ( 49 ). When we increased the radiation dose to 20 Gy, we observed a delayed tumor growth curve (results not shown) in the primary tumor, but this delay did not translate into long-term tumor control or elimination. Thus, it appears that low dose IGRT rather than high dose IGRT is preferable. Further improvements are possible with fractionated IGRT that affects tumor growth over a longer period. In a pilot study tracking CAR-T cell activity with luciferase after fractionated IGRT indicated the highest infiltration and expansion levels when tumors received four low daily doses of radiation. This insight highlights the need for further research to thoroughly understand the timing of CAR-T cell therapy after IGRT, with additional consideration of the radiation dosing schedules and possibility of multiple CAR-T cells treatments. Section title: Discussion Educational score: 4.151525974273682 Domain: biomedical Document type: Study Language: en Importantly, our study demonstrated that the combination of IGRT and anti-CEA scFab CAR-T therapy elicited not only a strong antitumor response, but also prevention of metastatic spread to the lungs. As a mechanism, we suggest that low dose IGRT at the primary tumor enhanced CAR-T cell infiltration and expansion in the primary tumor, allowing sufficient persistence and increased CAR T trafficking to distant metastatic sites as they developed over time. Section title: Discussion Educational score: 4.104499340057373 Domain: biomedical Document type: Study Language: en This study was limited to CAR-T therapy in immunocompromised animals, which restricts the ability to assess potential toxicity, an issue that we and other groups have previously shown in CEA transgenic mice treated with murine CAR T therapy without off-target effects ( 22 , 23 ). Another limitation is that a detailed cytokine panel analysis at different time points was not performed, leaving open the question of involvement of other activation markers aside from IFN-γ and granzyme B. The exhaustion markers analysis was limited to PD-1 and TIM3. However, the emergence of newer markers like Tox and CD39 in the measurement of T-cell exhaustion may better explain the observations of tumor escape ( 50 , 51 ). These aspects will be addressed in future experiments. Nevertheless, in our study the majority of the CD8+ CAR-T cells were PD-1 negative and expressed high levels of IFN-γ, both markers of effective CAR T therapy in clinical studies. Section title: Discussion Educational score: 4.0212202072143555 Domain: biomedical Document type: Study Language: en In summary, this study highlights the potential of anti-CEA scFab CAR-T cells as a promising therapeutic approach in combination with low dose IGRT. Building on our observations of the synergistic activity of anti-CEA scFab CAR-T cells with image-guided radiotherapy represents a novel therapeutic option that warrants clinical evaluation in solid tumor patients with metastases. | Review | biomedical | en | 0.999996 |
PMC11695364 | Section title: Introduction Educational score: 4.41792106628418 Domain: biomedical Document type: Review Language: en Gliomas are the most common primary brain tumors, characterized by high mortality and morbidity rates ( 1 ). According to the 2021 World Health Organization (WHO) Central Nervous System (CNS) classification, adult-type diffuse gliomas are categorized into astrocytomas (isocitrate dehydrogenase mutant [IDH-mt], 1p/19q non- codeletion), oligodendrogliomas (IDH-mt, 1p/19q codeletion), and glioblastomas (IDH wild-type, [IDH-wt]) ( 2 ). IDH-wt gliomas are classified as grade IV, oligodendrogliomas as grade II to III, and astrocytomas range from grade II to IV. Glioma grading influences treatment approaches, with high-grade gliomas typically managed by maximal surgical resection followed by adjuvant radiotherapy and chemotherapy, while low-grade gliomas are treated based on the extent of resection and patient factors such as age to determine postoperative adjuvant therapy ( 3 ). The new classification guidelines highlight the importance of genotypes and molecular characteristics. Research indicates that patients with 1p/19q codeletion respond better to radiotherapy and chemotherapy, resulting in improved prognosis ( 4 ). Additionally, O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation predicts a better response to temozolomide and enhances survival ( 5 ). However, molecular typing often relies on pathological diagnosis, which is invasive, prone to sampling errors, and costly. Section title: Introduction Educational score: 4.2702460289001465 Domain: biomedical Document type: Review Language: en MRI is the most commonly used preoperative diagnostic tool for gliomas. Grade IV gliomas frequently exhibit ring enhancement on T1-weighted images, whereas grade II and III gliomas typically show no enhancement, making it challenging to distinguish these grades on imaging. Most studies focus on the comparison between low-grade gliomas (grade II) and high-grade gliomas (grades III and IV), or between lower-grade gliomas (grades II and III) and higher-grade gliomas (grade IV), while the identification of grade III gliomas remains relatively vague. Diffusion-weighted imaging (DWI) has been used to predict MGMT promoter methylation and 1p/19q codeletion ( 5 , 6 ). However, apparent diffusion coefficient (ADC) measurements are often based on subjective regional delineation, and the heterogeneity of gliomas may introduce selection bias in region of interest settings. Some studies have found that the methylated MGMT promoter type exhibited larger ADC values, while others reported no differences between methylated and unmethylated types ( 7 , 8 ). Dynamic susceptibility contrast (DSC) and dynamic contrast-enhanced (DCE) imaging have also demonstrated value in predicting MGMT promoter methylation and 1p/19q codeletion, but both methods require contrast agent injection ( 9 – 12 ). Section title: Introduction Educational score: 4.101202964782715 Domain: biomedical Document type: Study Language: en The degree of tumor metabolism is often positively correlated with malignancy. High-grade gliomas exhibit vigorous cell proliferation, angiogenesis or vascular disruption, and an accumulation of more acidic metabolic byproducts in the extracellular space. Persistent hypoxia, increased glycolysis, and heightened acidity in tumors can affect tumor invasiveness and alter gene expression ( 13 , 14 ). Therefore, characterizing tumor metabolism and the acidity of the tumor microenvironment is a feasible method for grading and predicting molecular subtypes. Section title: Introduction Educational score: 4.693439960479736 Domain: biomedical Document type: Study Language: en Chemical exchange saturation transfer (CEST) imaging is an MRI technique that enhances the detection of low- concentration biomolecules by exploiting the chemical exchange properties between molecules and water protons ( 15 ). The exchange rates of certain protons are pH-dependent, making this technique useful for assessing tissue pH, which is crucial for evaluating the tumor microenvironment ( 16 ). Previous studies have demonstrated that CEST imaging of amine protons in glutamine molecules can be used as a noninvasive pH-weighted MRI technique for human and preclinical investigations of malignant gliomas ( 17 ). Amide proton transfer (APT) imaging is a relatively mature CEST technology, with amide protons in tissues serving as the primary source of the APT signal ( 18 ). Studies have shown that APT imaging holds potential for the differential diagnosis, grading, molecular typing, and prognostic evaluation of gliomas ( 19 – 24 ). However, APT imaging based on magnetization transfer asymmetry analysis can overlook confounding factors, including intrinsic semi-solid magnetization transfer (MT) asymmetry and low-field relayed nuclear Overhauser effect (NOE) signals. Methods such as multi-pool Lorentzian analysis and inverse Z-spectrum analysis have been proposed to enhance CEST quantitative analysis ( 25 , 26 ). Multi-pool Lorentzian analysis decomposes the Z-spectrum into five components: amide, NOE, amine, DS, and MT. Amide and amine represent mobile proteins/peptides and creatine, respectively. The DS signal is related to water proton concentration and tissue relaxation time, while the MT signal originates from immobile macromolecules. The NOE signal comes from the aliphatic and olefinic components of various metabolites, including mobile proteins, peptides, and lipids. Multi-pool Lorentzian analysis has demonstrated potential value in glioma grading and the diagnosis of IDH and 1p/19q genotypes ( 25 , 27 ). Section title: Introduction Educational score: 4.101353645324707 Domain: biomedical Document type: Study Language: en In this study, we quantitatively describe glioma metabolism and the pH characteristics of the tumor region based on CEST imaging using multi-pool Lorentzian and pH analyses. We explore their value in assessing glioma grade, IDH mutation, 1p/19q codeletion, and MGMT promoter methylation. Additionally, histogram analysis was employed to better characterize tumor heterogeneity. Section title: Patient cohort Educational score: 3.5608808994293213 Domain: biomedical Document type: Study Language: en This study received approval from the Institutional Review Board. Between January 2023 and March 2024, 415 consecutive patients suspected of having gliomas who underwent preoperative CEST MRI examinations were enrolled. The inclusion criteria were: (1) histologically diagnosed adult-type diffuse gliomas, and (2) age >18 years. Figure 1 illustrates the participant flowchart. Section title: Data acquisition Educational score: 4.210804462432861 Domain: biomedical Document type: Study Language: en Scans were performed using a 3TIngenia CX Philips scanner with an 80 mT/mgradient, 200 mT/m/s slew rate, and a 32-channel head coil. Routine structural MRI included T1-weighted images before and after Gd enhancement, and T2-FLAIR images, with a total acquisition time of 10 minutes. A custom-developed CEST sequence based on 2D multi-offset, single-slice, single-shot turbo spin echo (TSE) was applied to the maximum cross-sectional areas of the tumors with the following acquisition parameters: radiofrequency (RF) saturation power, 0.9 µT; saturation duration, 3,000 ms; TR = 5000 ms, TE = 14 ms, field of view = 200 × 200 mm 2 , voxel of 2.5 × 2.5 × 4 mm 3 , compressed sensing acceleration factor of 4, and flip angle 90 degrees. RF saturation was performed with 2 parallel RF transmission channels (through a body coil) driven by the RF amplifiers in a time‐interleaved fashion. By combining 2 amplifiers, each operating at 50% duty cycle, RF saturation at 100% duty cycle was achieved. The 64 offsets in order were 0, ± 0.25, ± 0.5, ± 0.75, ± 1, ± 1.25, ± 1.5, ± 1.75, ± 2, ±2.25, ± 2.5, ± 2.75, ± 3, ± 3.25, ± 3.5, ± 3.75, ± 4, ± 4.25, ± 4.5, ± 4.75, ± 5, ± 5.5, ± 6, ± 6.5, ± 7, ± 7.5, ± 10, ± 15, ± 20, ± 25, ± 30, ± 100 and +300 parts per million (ppm).The scan duration was 5 minutes and 25 seconds. Section title: Image analysis Educational score: 4.141824722290039 Domain: biomedical Document type: Study Language: en The tumor region-of-interest (ROI) was manually delineated by two dedicated radiologists (with 3 and 10 years of neuroradiology experience, respectively) on CEST images. Areas with necrosis, cysts, and hemorrhages were carefully excluded. The solid tumor was defined as either the contrast-enhanced region on T1-weighted images or the hyperintense region on T2-FLAIR images (when contrast enhancement was not detected) ( 25 , 28 , 29 ). Based on a previous study ( 30 ), we used custom MATLAB code for quantitative image analysis of CEST. The Z-spectrum is generated using the ratio between the saturated image Ssat and the fully relaxed image S0. The spline-interpolated Z-spectrum was used to calculate frequency differences for generating B0 maps and performing voxel-wise B0 field correction. The B0-corrected Z-spectrum was then fitted as a sum of five Lorentzian functions corresponding to aliphatic nuclear Overhauser effect (NOE, -3.5 ppm), magnetization transfer (MT, -1 ppm), direct saturation of water (DS, 0 ppm), amine (2.0 ppm), and amide (3.5 ppm). Section title: Image analysis Educational score: 4.152554988861084 Domain: biomedical Document type: Study Language: en The spectrally selective CEST effects were obtained through Lorentzian line fitting for four steps: (1) motion correction using a subpixel image registration algorithms and denoising raw images using multilinear singular value decomposition; (2) 2-pool Lorentzian fitting (MT and DS) for B0 determination and B0 correction (3) 2-pool Lorentzian fitting (MT and DS) on B0 corrected data, generation of MTRLD; (4) 3-pool Lorentzian model fitting of MTRLD for isolated CEST contrast. Section title: Image analysis Educational score: 4.1352081298828125 Domain: biomedical Document type: Study Language: en The first step involved utilizing a 2-pool model to characterize background signals such as direct water saturation (DS) and semisolid magnetization transfer (MT). Only those irradiation frequency offsets, assumed to be influenced exclusively by the background signal, were employed for the fit (MT: ± 10, ± 15, ± 20, ± 25, ± 30, ± 100; water: ± 1, ± 0.75, ± 0.5, ± 0.25, 0 ppm). Any other irradiation frequency offsets were disregarded. The 2-pool fit model used is expressed by the DS(w) and MT. Section title: Image analysis Educational score: 3.1545310020446777 Domain: biomedical Document type: Study Language: en with a constant c and the adjusted Lorentzian Lw of the water line. Lw includes a plateau to account for the pulse bandwidth at 3T defined by Equation 2 . Section title: Image analysis Educational score: 4.235311985015869 Domain: biomedical Document type: Study Language: en where A represents the Lorentzian amplitude of the five pools, Γ represents the Lorentzian width (full-width-at-half-maximum) of the five pools, and δ represents the peak position. Here, Θ[•] refers to the Heaviside function, with x = ( Δ ω − δ ω − B W 2 ) and y = ( Δ ω − δ ω + B W 2 ) . The parameter BW is an estimate of the Fourier width of the Gaussian saturation pulse, which is related to platform width and remains constant for B W = 1 t p u l s e γ 2 π . The second pool in which the Lorentzian function is defined in Equation 3 represents MT: Section title: Image analysis Educational score: 4.07780647277832 Domain: biomedical Document type: Study Language: en The Lorentzian ssMT pool was fitted with an initial resonance frequency of -1 ppm, which was adjustable within the range from 0 to -2.5 ppm during data fitting ( 30 ) In the second step, the water pool’s off-resonance in the preliminary 2-pool model served as a surrogate B0 map. Z-spectra underwent shifts to compensate B0 inhomogeneity. Section title: Image analysis Educational score: 3.026000738143921 Domain: biomedical Document type: Study Language: en In accordance with prior research, the Lorentzian difference method was employed for the evaluation of peak-selective CEST. Section title: Image analysis Educational score: 3.142707586288452 Domain: biomedical Document type: Study Language: en In step 3, the Z f i t , r e f referred to a 2-pool background fit, which was repeated on B0-corrected and denoised Z-spectra. Section title: Image analysis Educational score: 4.092546463012695 Domain: biomedical Document type: Study Language: en Ultimately, in the step 4, a 3-pool Lorentzian model was implemented to fit the MTR LD spectrum to distinctly separate the amide (+3.5 ppm), amine (+2.0 ppm), and NOE (-3.5 ppm) resonances. Section title: Image analysis Educational score: 1.469368577003479 Domain: biomedical Document type: Other Language: en and ( Equation 6 ) Section title: Image analysis Educational score: 3.5086820125579834 Domain: biomedical Document type: Study Language: en Quantitative maps were derived from the fitting parameter Ax for the five CEST pools. Section title: Image analysis Educational score: 3.202059030532837 Domain: biomedical Document type: Study Language: en The conventional magnetization transfer ratio (MTR) asymmetry analysis was used to calculate the MTR 3.5 , defined as Section title: Image analysis Educational score: 2.751133441925049 Domain: biomedical Document type: Study Language: en We use CEST to characterize the acidity of the tumor region according to ( 17 ). Section title: Image analysis Educational score: 3.533250570297241 Domain: biomedical Document type: Study Language: en At last, the histogram values for various parameters such as amide, NOE, amine, MT, DS, MTR 3.5 , and pH_weighted ( 13 , 17 ) in tumor were calculated. Section title: Statistical analysis Educational score: 4.059072017669678 Domain: biomedical Document type: Study Language: en Data were analyzed using SPSS 25.0, GraphPad Prism version 8.0, and MedCalc 20.0. The inter-observer variability of measurements in glioma patients was assessed using the intra-class correlation coefficient. Continuous variables with normal distribution were expressed as the mean ± SD, while non-normally distributed variables were expressed as the median with IQR. Categorical variables were expressed as frequencies. Metrics with significant differences were identified using an independent sample t-test (for normally distributed data) or Mann–Whitney U test (for non-normally distributed data). The Chi-squared test was used for categorical variables. Among the histogram features of amide, NOE, amine, MT, DS, pH_weighted, and MTR3.5, those with statistical significance (p < 0.05) were first selected. Features demonstrating the highest diagnostic performance were further selected. Collinearity analysis was performed on these features, and those with a tolerance (Tol) less than 0.1 or a variance inflation factor (VIF) greater than 10 were excluded. The remaining features were retained for constructing the combined model. Individual features or combined models were used for glioma grading and molecular typing (IDH mutation, 1p/19q codeletion, and MGMT promoter methylation status). The significance level was set at p = 0.05 for all tests. Section title: Patient information Educational score: 4.110747814178467 Domain: biomedical Document type: Study Language: en The demographic and pathological findings of the participants are summarized in Table 1 . A total of 128 patients with histologically confirmed gliomas were included. There were 24 grade II, 16 grade III, and 88 grade IV gliomas, among which 82 were IDH-wt and 46 were IDH-mut. Significant differences were found in age across different grades (p = 0.018) and IDH subtypes (p = 0.014). Among grade II and III gliomas, there were 18 and 22 with 1p/19q codeletion, respectively, and the remaining without codeletion. No significant differences in age were observed between these groups (p = 0.667, 0.683, respectively). There were 17 gliomas with MGMT promoter methylation and 16 without. Patients with MGMT promoter methylation were significantly older than those without (p = 0.043). No significant differences were found in gender across all subgroups (p = 0.785, 0.963, 0.214, 0.315, 0.728, respectively). We performed an inter-observer consistency analysis for all gliomas, low-grade gliomas, and high-grade gliomas separately, and the results showed good consistency in both groups. The intraclass correlation coefficients for inter-observer agreement for CEST metric values ranged from 0.90 to 0.99( Supplementary Tables S1 – S3 ). Section title: CEST metrics in distinguishing grade II and grade III gliomas Educational score: 4.10754919052124 Domain: biomedical Document type: Study Language: en As shown in Table 2 and Figure 2 , grade III gliomas exhibited higher DS (median: 0.80 vs 0.78, p = 0.020) and pH_weighted (median: -0.01 vs -0.02, p = 0.008) signals, and lower MT (mean: 0.14 vs 0.15, p = 0.029) compared to grade II gliomas. Specifically, the 90th percentile of MT [p = 0.003, AUC = 0.78 (95% CI: 0.62- 0.90)], the mean of DS [p = 0.020, AUC = 0.72 (95% CI: 0.55-0.85)], and the mean of pH_weighted [p = 0.006, AUC = 0.76 (95% CI: 0.59-0.88)] showed the best performance for each signal, respectively . The combined model achieved an AUC of 0.80 (95% CI: 0.64-0.91) ( Supplementary Table S4 ). Section title: CEST metrics in distinguishing grade III and grade IV gliomas Educational score: 4.119319438934326 Domain: biomedical Document type: Study Language: en As shown in Table 2 and Figure 2 , grade IV gliomas exhibited higher amide (mean: 0.06 vs 0.04, p = 0.001) , NOE (mean: 0.06 vs 0.05, p = 0.034), MT (mean: 0.16 vs 0.14, p = 0.031), pH_weighted (75th pc: 0.008 vs -0.003, p = 0.037), and lower DS (mean: 0.78 vs 0.80, p = 0.013) compared to grade III gliomas. Specifically, the 75th percentile of amide [p < 0.001, AUC = 0.78 (95% CI: 0.69-0.86)], the 75th percentile of NOE [p = 0.017, AUC = 0.69 (95% CI: 0.59-0.78)], the 90th percentile of MT [p = 0.018, AUC = 0.69 (95% CI: 0.59-0.78)], the 25th percentile of DS [p = 0.009, AUC = 0.71 (95% CI: 0.61-0.79)], and the 75th percentile of pH_weighted [p = 0.037, AUC = 0.67 (95% CI: 0.57-0.75)] showed the best performance for each signal, respectively . The combined model achieved an AUC of 0.83 (95% CI: 0.74-0.90) ( Supplementary Table S4 ). Section title: CEST metrics in distinguishing IDH wide type and mutant type gliomas Educational score: 4.12401008605957 Domain: biomedical Document type: Study Language: en As shown in Table 4 , Figure 4 , IDH-wt gliomas exhibited higher amide (mean: 0.06 vs 0.04, p < 0.001) , NOE (mean: 0.06 vs 0.07, p = 0.031), pH_weighted (mean: -0.002 vs -0.009, p < 0.001), and MTR3.5 (mean: 0.003 vs -0.010, p = 0.009) compared to IDH-mut gliomas. Specifically, the median of amide [p < 0.001, AUC = 0.83 (95% CI: 0.75-0.89)], the 10th percentile of NOE [p = 0.013, AUC = 0.64 (95% CI: 0.55-0.72)], the median of pH_weighted [p < 0.001, AUC = 0.76 (95% CI: 0.68-0.83)] and the 25th percentile of MTR3.5 [p = 0.002, AUC = 0.67 (95% CI: 0.58-0.75)] performed the best for each type of signal, respectively . The combined model achieved an AUC of 0.84 (95% CI: 0.77-0.90) ( Supplementary Table S4 ). Section title: CEST metrics in detecting 1p19q codeletion status Educational score: 4.099384307861328 Domain: biomedical Document type: Study Language: en As shown in Table 5 and Figures 4 , 5 , for grade II gliomas, 1p/19q non-codeletion gliomas exhibited higher MT compared to 1p/19q codeletion gliomas (mean: 0.17 vs 0.14, p = 0.007) . The 90th percentile of MT achieved the best performance [p = 0.003, AUC = 0.87 (95% CI: 0.65-0.98)] . For grade III gliomas, 1p/19q non-codeletion gliomas exhibited higher pH_weighted signals compared to 1p/19q codeletion gliomas (mean: -0.004 vs -0.013, p = 0.038) . The 25th percentile of pH_weighted achieved the best performance [p = 0.028, AUC = 0.83 (95% CI: 0.56-0.97)] . Section title: CEST metrics in detecting MGMT promoter methylation status Educational score: 4.090730667114258 Domain: biomedical Document type: Study Language: en As shown in Table 5 and Figure 6 , MGMT promoter unmethylated gliomas exhibited higher MTR3.5 signals compared to MGMT promoter methylated gliomas (mean: 0.01 vs -0.01, p = 0.048) . The 90th percentile of MTR3.5 achieved the best performance [p = 0.005, AUC = 0.79 (95% CI: 0.61-0.91)] . Section title: Discussion Educational score: 4.282278537750244 Domain: biomedical Document type: Study Language: en This study investigated glioma grading and molecular genotyping using CEST-based pH assessment and micro- metabolic profiling within the context of the 2021 WHO CNS classification. Our results indicate that multi-pool Lorentzian analysis and pH-weighted analysis demonstrate diagnostic performance in grading gliomas and ingenotyping for IDH mutation status, 1p/19q co-deletion, and MGMT promoter methylation status.pH_weighted imaging can characterize the acidic microenvironment of tumors. We found that high-grade gliomas, IDH-wt gliomas, and 1p/19q non-codeleted gliomas exhibited higher pH_weighted values compared to low-grade gliomas, IDH-mt gliomas, and 1p/19q codeleted gliomas. Tumor cells preferentially convert glucose to lactic acid even in the presence of oxygen, resulting in excessive lactic acid production. Additionally, poor vascularization in these tumors leads to hypoxic conditions that further drive glycolysis and acid production ( 15 ). The higher metabolic activity in more invasive gliomas results in hypoxia and the accumulation of acidic metabolic products, leading to larger pH_weighted values ( 31 ). As shown in Figure 5 , gliomas with and without 1p/19q codeletion are difficult to differentiate on T2-FLAIR and T1-enhancement images. However, pH_weighted imaging can visually highlight differences between them and better reflect tumor heterogeneity. In the central tumor region, acidity is significantly increased, while in the peritumoral edema zone, tumor acidity is relatively lower. In more invasive IDH-wt gliomas, both the central tumor region and the peritumoral edema zone exhibit higher acidity, partially explaining their greater invasiveness. Therefore, we believe that pH_weighted imaging is a promising biomarker for glioma grading and subtyping analysis. Section title: Discussion Educational score: 4.438848495483398 Domain: biomedical Document type: Study Language: en MT and DS respectively represent the content of semi-solid molecular tissue and water molecules. Grade III gliomas exhibited higher DS and lower MT compared to grade II gliomas. DS is related to tissue water proton density. Research indicates that high-grade gliomas tend to have higher vascular endothelial growth factor (VEGF) expression ( 32 ). VEGF is known as a potent growth factor for vascular endothelial cells, playing a crucial role in tumor growth and invasion by promoting the proliferation and migration of tumor vascular endothelial cells, increasing tumor vascular permeability, and inducing tumor lymphangiogenesis ( 32 , 33 ). The higher VEGF expression corresponds with more severe edema, resulting in higher DS ( 25 ). However, our findings showed that DS was lower in grade IV gliomas compared to grade III gliomas, possibly due to differences in ROI selection. In grade III gliomas, where most cases did not show enhancement on T1-weighted images, the entire T2 hyperintense region was selected as the ROI. In contrast, grade IV gliomas were characterized by selecting the enhanced T1 area and excluding the peritumoral edema zone. MT primarily originates from immobile macromolecules, such as proteins and polysaccharides, and may serve as an indicator of white matter integrity ( 34 ). MT was higher in grade II gliomas, likely due to their retention of more normal brain tissue structure and composition. In grade IV gliomas, elevated cell density may lead to increased levels of proteins, polysaccharides, and other components within the tumor region, resulting in a higher MT effect. Furthermore, MT was associated with 1p/19q codeletion in grade II gliomas, with 1p/19q codeletion gliomas showing lower MT . Further subclassification of 1p/19q codeletion and non-codeletion within low-grade gliomas is meaningful, as the identification of 1p/19q codeletion in IDH-mt gliomas maybe influenced by histological grading. Distinguishing 1p/19q subtypes in grade II/III gliomas can facilitate more precise treatment planning and efficacy assessment in preoperative or postoperative follow-up. Section title: Discussion Educational score: 4.174535274505615 Domain: biomedical Document type: Study Language: en Amide and NOE reflect the content of amide protons and macromolecules such as lipids within the tissue. Significant differences were observed in amide and NOE signals between grade III and IV gliomas, as well as between IDH-wt and IDH-mt gliomas. IDH-wt gliomas are typically more aggressive and have higher cellular density compared to IDH-mt gliomas. This aggressive phenotype is associated with an increased proliferative rate and elevated protein synthesis ( 23 , 35 ). In contrast, IDH-mt leads to the production of the oncometabolite 2- hydroxyglutarate (2-HG), which results in abnormal methylation of DNA and histones, affecting gene expression and cell differentiation ( 36 ). The higher concentration of proteins and peptides in IDH-wt gliomas likely contributes to a stronger amide and NOE signal. We found that the diagnostic performance of amide is superior to MTR 3.5 . This maybe because MTR 3.5 is influenced by signals such as NOE and DS, and therefore cannot reflect a purer source of the amide signal. Section title: Discussion Educational score: 4.122114181518555 Domain: biomedical Document type: Study Language: en Our study also found that MGMT promoter unmethylated gliomas typically exhibit higher amide signals. MGMT promoter methylation in gliomas is associated with reduced protein expression, which may impact the expression of downstream proteins. Therefore, CEST may serve as a useful imaging biomarker for predicting MGMT methylation status, consistent with previous findings ( 37 ). In contrast to previous studies where APT could not predict MGMT promoter methylation, possibly due to smaller sample sizes ( 26 ), our results suggest that MTR 3.5 , despite being affected by multiple factors, can predict MGMT promoter methylation more effectively than the relatively pure amide signals. Section title: Discussion Educational score: 4.2136969566345215 Domain: biomedical Document type: Study Language: en In our study, the amine signal showed no significant differences in the grading and molecular classification of gliomas. Notably, Zhu et al.’s research also found no differences in the amine signal between IDH wild-type and mutant gliomas ( 27 ). We believe that there are two possible reasons for this result. Firstly, the amine signal has been assumed to mainly represent the contribution from creatine amine protons. However, amine signal obtained through Lorentzian fitting frequently overlaps with other rapidly exchanging pools, like glutamate, making it difficult to isolate them under 3T conditions. Creatine provides phosphate through phospho-creatine for adenosine triphosphate synthesis in the cell energy requirement. Tumor has reduced creatine and tumor creatine further reduces with tumor progression presumably due to elevated energy deficiency ( 38 ). There is building evidence that alterations to glutamate homeostasis in gliomas play an important role in diffuse glioma cell survival and increased extra-cellular glutamate causes excitotoxicity to peri-tumoral structures and promotes tumor invasion in pre-clinical studies ( 39 ). The complex variations in the contents of various components within the tumor lead to fluctuations in the amine signal. Secondly, our study was conducted using a 3T MRI scanner. Due to the relatively fast exchange rate of the amine signal and its fitting being close to the water peak at 2 ppm, the z-spectrum characteristics may not be distinct enough, making the Lorentzian fitting more challenging. In summary, the amine signal in glioma research may be influenced by various factors. Further research may need to explore more sensitive techniques or methods to better understand the role of the amine signal in gliomas. Based on the above discussion, the combination of multi-pool Lorentz analysis and pH analysis based on CEST demonstrates good performance in improving the grading and IDH gene typing of glioma. MT and pH_weighted can effectively identify 1p/19q codeletion in grade II and grade III gliomas, respectively. MTR 3.5 demonstrates potential effect in identifying MGMT promoter methylation. This technology can be implemented on standard MRI equipment, with a scanning time of approximately 5 minutes being clinically feasible. It does not require additional injection of contrast agents, making it relatively safe. In our study, we chose to perform the scans before the injection of the contrast agent to avoid the influence of the contrast agent on the CEST effect ( 40 ). Section title: Discussion Educational score: 4.292271614074707 Domain: biomedical Document type: Study Language: en However, our study has several limitations. Firstly, although existing literature has demonstrated that, within lower irradiation power ranges, multi-pool Lorentzian fitting offers superior quantification accuracy compared to the three-frequency offset method and the Lorentzian-Dipolar (LD) method ( 38 ). However, multi-pool Lorentzian fitting has several limitations. In situations where the resonance frequencies of different signals are closely spaced or mixed, Lorentzian fitting may struggle to effectively differentiate between these signals. For example, the wide ‘MT’ peak could have multiple contributions especially the NOE(-1.6), which have attracted many interests in recent years ( 41 – 44 ). However, we could not resolve these components precisely in our analysis. Such spectral overlap can lead to inaccuracies in the fitting results, adversely impacting the quantification of specific signals, such as those from amines or other metabolites ( 27 ). Besides, Lorentzian fitting exhibits high sensitivity to background noise, particularly when signal intensities are low. The presence of background noise can interfere with the fitting process, resulting in erroneous parameter estimates. Additionally, successful Lorentzian fitting requires careful selection of initial parameters and fitting ranges. Inappropriate parameter choices can lead to convergence on local minima, thus compromising the accuracy and reliability of the fitting results. These limitations underscore the importance of judiciously selecting appropriate fitting methods and parameters in practical applications to ensure the reliability and validity of the results. Secondly, for pH assessment, although research indicates that amine proton-based CEST imaging (with a resonance frequency of approximately 3.0 ppm) can provide pH-weighted image contrast and may serve as an important imaging biomarker for human brain gliomas ( 17 ). The measured CEST contrast depends on various technical factors, including the shape, duration, length, amplitude and repetition time of the saturation pulse, and the strength of the scanning field, and the concentration of amine protons. Additionally, the image SNR can affect pH measurements ( 45 ). Furthermore, exchangeable protons from other proteins or macromolecules within the tissue may also influence the amine signal ( 27 ).Further research is needed to standardize CEST scanning protocols and post-processing techniques to optimize signal acquisition and data fitting. Additionally, larger-scale clinical studies are required to investigate pH variations among different tumor grades and molecular subtypes across the entire tumor. Thirdly, the sample size is relatively small, particularly for the 1p/19q expression status subgroup. Further validation in a larger cohort is necessary. As a single-center study, there are inherent limitations such as reduced generalizability and potential biases. Multi-center studies are needed to validate and expand upon these findings. Lastly, due to time constraints, only 2D single-slice imaging was performed, which might have missed important pathological regions due to intra-tumoral heterogeneity. Implementing 3D acquisition to cover the entire tumor could address this issue. Section title: Conclusion Educational score: 4.063529014587402 Domain: biomedical Document type: Study Language: en In summary, our findings indicate that quantitative assessment of tumor metabolism and microenvironment acidity through multi-pool Lorentzian analysis and pH-weighted analysis can serve as indicators for glioma grading, and for predicting IDH mutations, 1p/19q codeletion, and MGMT promoter methylation status. These metrics not only provide valuable insights into tumor subgroups but also reflect the heterogeneity within tumors. | Review | biomedical | en | 0.999995 |
PMC11695366 | Section title: Introduction Educational score: 4.037195205688477 Domain: biomedical Document type: Review Language: en Neurodevelopmental disorders(NDDs) are a group of chronic developmental brain disorders, including autism spectrum disorder(ASD), intellectual developmental disorder (IDD), developmental speech or language disorder (DLD), attention-deficit/hyperactivity disorder (ADHD), and tic disorders . The prevalence of NDDs among individuals under the age of 18 years, as defined by the Diagnostic and Statistical Manual of Mental Disorders , Fifth Edition (DSM-5) criteria, has been infrequently measured. Existing studies have reported the following prevalence rates: ASD has a prevalence rate ranging from 0.70 to 3% of the population, ID occurs at a rate of 0.63%, ADHD has a prevalence rate ranging from 5 to 11%, among other conditions . The pathogenesis of NDDs is not fully understood; however, it is mainly caused by genetic and neurobiological factors and shows a trend of continuous development . Section title: Introduction Educational score: 4.883387565612793 Domain: biomedical Document type: Study Language: en Collapsin response mediator proteins (CRMPs), also known as dihydropyrimidinase-like (DPYSL) proteins, members of the cytoplasmic phosphoprotein family, consist of five cytoplasmic phosphoproteins (CRMP1-5) that are widely expressed in the nervous system and are essential for brain development and function. They play crucial roles in cellular processes, such as cell migration, neurite extension, axonal guidance, dendritic spine development, and synaptic plasticity, through their phosphorylation status . CRMP2, the first protein of the CRMP family to be cloned, was identified as an intracellular messenger required for Sema3A-induced growth cone collapse . It has also been shown that CRMP3-deficient ( CRMP3 −/− ) mice exhibit abnormal dendritic fluctuations, confirming the importance of CRMP3 in hippocampal dendritic organization . CRMP1, also known as UNC-33-like phosphoprotein 1 (ULIP1), is involved in signaling protein-induced growth cone collapse during neurodevelopment, as part of the protein signal transduction pathway in the brain. CRMP1 is involved in the dendritic development of cortical pyramidal neurons, and it also mediates the transmission of the reelin signal in cortical neuron migration and regulates the migration of cerebral cortical neurons . Given their key function in developmental processes, disturbances in CRMP function can result in neurodevelopmental diseases . Section title: Introduction Educational score: 4.336627960205078 Domain: biomedical Document type: Study Language: en At present, there are few reports on CRMP1 and human neuropsychiatric disorders. In chronic brain diseases, such as Alzheimer’s disease and Parkinson’s disease, pathological features include misfolding or abnormal aggregation of various proteins, including CRMP1 . In 2012, Verian Bader et al. demonstrated that lymphoblastic cell lines from patients with schizophrenia showed increased CRMP1 expression . Researchers have also found that maternal autoantibody reactivity to CRMP1 is associated with the severity of ASD . Da Silva et al. reported that a partial coding sequence of CRMP1 overlaps with the Ellis-van Creveld (EVC) gene, identifying CRMP1 as a potential genetic modifier of the severity of EVC syndrome, with approximately 10% of EVC patients possibly exhibiting neurodevelopmental abnormalities . To date, only one report has linked genetic variations in the CRMP1 gene to neurodevelopmental disorders in humans. This report described a heterozygous de novo mutation in the CRMP1 gene in three unrelated individuals with muscular hypotonia, intellectual impairment, and/or autism spectrum disorder. This mutation is believed to cause neurodevelopmental disorders that affect protein oligomerization and neurite growth . Section title: Introduction Educational score: 3.858558416366577 Domain: biomedical Document type: Clinical case Language: en We present a case of a boy with a heterozygous de novo variant in the CRMP1 gene, identified through whole-exome sequencing (WES), who exhibits phenotypes of a neurodevelopmental disorder such as autism, language delay, hyperactivity, and learning disabilities. Section title: Case presentation Educational score: 2.179053544998169 Domain: clinical Document type: Clinical case Language: en A 9-year-old boy was referred to the pediatric behavioral development clinic due to his poor verbal expression and tendency to play alone. He is the first child born to a healthy, non-related couple after a normal pregnancy. He was delivered via cesarean section at full term, with a birth weight of 3,600 g. He was able to walk independently at 15 months old and began speaking at approximately 24 months old. Section title: Case presentation Educational score: 2.1674447059631348 Domain: clinical Document type: Clinical case Language: en During kindergarten, he was able to express some ideas using short sentences and understand some common-sense questions, but he had poor eye contact, was not sociable, and lacked awareness of danger. At 7 years of age, he was socially impaired and inattentive. In the hospital, the individual’s Full Scale Intelligence Quotient (FSIQ), as assessed by the Wechsler Intelligence Scale for Children (WISC), was found to be 69, indicating that his cognitive abilities are below the average range for his age group. The conditions mentioned did not improve after the age of 9 years and were characterized by academic deficiency, reduced attention to surroundings, and a limited range of interests, stereotypic behavior, and hypersensitivity to sound or touch. Section title: Case presentation Educational score: 4.058774948120117 Domain: clinical Document type: Clinical case Language: en He came to our hospital for treatment, and the Autism Diagnostic Observation Schedule (ADOS) was used for evaluation. The scores were as follows: 10 points for Social Affect (SR) and one point for Restricted and Repetitive Behaviors (RRB), with a total score above the autism spectrum threshold of seven points. The Autism Diagnostic Interview-Revised (ADI-R) yielded the following scores: 16 points in the Reciprocal Social Interactions domain, 14 points in the Language/Communication domain, and two points in the Restricted, Repetitive, and Stereotyped Behaviors and Interests domain. These scores suggested abnormal communication and the presence of stereotypic behaviors. In the Diagnostic Receptive and Expressive Assessment of Mandarin–Comprehensive (DREAM-C), the individual scored 76 points in the expressive language domain, which was below the normative level of expressive language for his peers. In addition, the FSIQ, as assessed by the WISC, was found to be less than 40, suggesting mild delay. His 25-hydroxyvitamin D3 level was 16.21 ng/mL, and routine metabolic screening showed abnormal results. Section title: Whole-exome sequencing Educational score: 4.136215686798096 Domain: biomedical Document type: Study Language: en DNA was obtained from the peripheral blood of the patient and his parents. The DNA was then submitted for trio (proband and parents) whole-exome sequencing (trioWES) to Chigene (Beijing) Translational Medical Research Center Co. Ltd. Protein-coding exome enrichment was performed using xGen Exome Research Panel v2.0 (IDT, Iowa, United States), which consists of 429,826 individually synthesized and quality-controlled probes. This panel targets 39 Mb of the protein-coding region (19,396 genes) of the human genome and covers 51 Mb of end-to-end tiled probe space. High-throughput sequencing was performed using the MGISEQ-T7 series sequencer, with atleast 99% of the target sequence being sequenced. The sequencing process was conducted by Chigene (Beijing) Translational Medical Research Center Co. Ltd. Section title: Results Educational score: 3.9299519062042236 Domain: biomedical Document type: Study Language: en After obtaining informed consent from the family, genetic testing was conducted. Through WES, a frameshift variant was identified in the proband: NM 001014809.3 (CRMP1) :c.1755del (p.Lys586fs). The proband is heterozygous for this variant, and both parents are wild-type, indicating that it is a de novo mutation. We submitted this mutation to ClinVar . The variant was confirmed by Sanger sequencing . Section title: Results Educational score: 4.296010494232178 Domain: biomedical Document type: Study Language: en The CRMP1 gene contains 14 exons and encodes a protein of 686 amino acids . According to the Pfam database, this protein contains an Amidohydro_1 domain, which belongs to the amidohydrolase family. Amidohydro_1 is a large superfamily of metal-dependent hydrolases . Ravindran et al. reported three missense variations in the CRMP1 gene, two of which [NM_001014809.3 ( CRMP1 ): c.1280C > T (p.T427M) and NM_001014809.3 ( CRMP1 ):c.1052 T > C (p.F351S)] are located within this structure, while the other variation, NM_001014809.3 ( CRMP1 ):c.1766C > T (p.P589L), is located downstream of this structure . Section title: Discussion Educational score: 3.6711373329162598 Domain: biomedical Document type: Study Language: en The frameshift mutation identified in this report (p.Lys586fs) was located near the p.P589L site. Section title: Discussion Educational score: 4.073589324951172 Domain: biomedical Document type: Study Language: en Neurons are the fundamental structural and functional units of the nervous system, consisting of the soma, membrane, cytoskeleton, dendrites, and axons, and are responsible for information processing and signal transmission. Neural circuit development and adaptability are crucial for nervous system function. CRMPs, highly expressed in the developing nervous system, are integral to neural development, influencing axon guidance, synaptic maturation, cell migration, and adult brain function . Section title: Discussion Educational score: 4.96461820602417 Domain: biomedical Document type: Study Language: en CRMP1 is an intracellular mediator of Sema3A signaling, an important molecule that directs axon outgrowth and branching. By binding to microtubules and F-actin, CRMP1 regulates cytoskeletal depolymerization and reorganization, thereby affecting growth cone movement and orientation . CRMP1 also plays an important role in synapse formation and maturation. It promotes efficient connectivity between neurons by regulating the organization and function of both presynaptic and postsynaptic components . Phosphorylation of CRMP1 by Fyn kinase at Y504 is essential for neuronal migration, regulating growth cone behavior, and axon pathfinding . As a cyclin-dependent kinase 5 (Cdk5) substrate, CRMP1 is vital for actin cytoskeleton remodeling, which is critical for neuronal development and synaptic plasticity . The phosphorylation of CRMP1 by kinases such as Cdk5, glycogen synthase kinase-3β (GSK-3β), and Rho-associated kinase (ROCK) alters its conformation, stability, and protein interactions, thereby impacting microtubule dynamics and neuronal morphogenesis . In addition, CRMP1’s localization to the midbody during cytokinesis suggests that it may play a role in the midbody query-query PPI (QQ-PPI) network, influencing the process of abscission and termination of cytokinesis . Section title: Discussion Educational score: 4.071316242218018 Domain: biomedical Document type: Review Language: en In summary, CRMP1 is a multifaceted protein essential for the development and maintenance of the nervous system. Its roles in axon guidance, dendritic development, neurite outgrowth, and neuronal migration make it a critical factor in the complex orchestration of neurodevelopment. Disruptions in CRMP1’s function, such as those caused by genetic variants, can lead to neurodevelopmental disorders, highlighting the importance of CRMP1 in brain health and function. Section title: Discussion Educational score: 3.4099619388580322 Domain: biomedical Document type: Study Language: en We searched the PubMed and Web of Science databases, and found, as mentioned earlier, the only article on the association between CRMP1 and human neurodevelopmental disorders. Through the bioinformatics analysis of three CRMP1 cases and the analysis of the effect of genetic variants on protein structure, it was emphasized that CRMP1 is related to human neurodevelopmental disorders . Section title: Discussion Educational score: 3.9857141971588135 Domain: biomedical Document type: Study Language: en As shown in Table 1 , we summarized some common or different characteristics of these four probands. The head MRI of the four cases was normal. Although the cranial MRI findings of the four patients mentioned were within normal limits, recent studies have indicated that MRI remains a beneficial supplementary diagnostic tool in the evaluation of patients with neurodevelopmental disorders (NDDs) of obscure origin. It is especially useful for patients presenting with neurological symptoms or signs, such as motor dysfunction, pyramidal tract disorders, epilepsy, or abnormal head circumference. After conventional metabolic and genetic assessments yield normal results, the diagnostic value of MRI is heightened in these patients as it has the potential to reveal structural brain anomalies that either support or lead to an etiological diagnosis of NDDs . Section title: Discussion Educational score: 4.232099533081055 Domain: biomedical Document type: Study Language: en They all had speech and language delays, as well as behavioral problems. Both proband 1 (P1) and proband 3 (P3) were diagnosed with autism. For children with neurodevelopmental disorders, dynamic changes may occur in those with and without ASD. It has been confirmed through a comprehensive behavioral test battery on CRMP1 knockout ( crmp1 −/−) mice that these mice exhibited hyperactivity, impaired context-dependent memory, and deficits in long-term memory retention . CRMP1 is one of the proteins targeted by maternal autoantibodies in a subset of mothers with children diagnosed with ASD. The presence of these autoantibodies, particularly against CRMP1, has been associated with more severe ASD symptoms, as measured by the ADOS scores . SEMA3A is crucial for neurons during the first 13 weeks of pregnancy. CRMP1 is one of the sensors of SEMA3A signaling, and its mutation may affect the normal growth and differentiation of neurons and is also related to ASD . Section title: Discussion Educational score: 4.130539894104004 Domain: biomedical Document type: Study Language: en Interestingly, P2–P4 were reported to have bradykinesia. CRMP1 phosphorylation has been shown to affect motor function in mice. Inhibition of CRMP1 phosphorylation at Ser522, achieved through the use of Crmp1 ki/ki mice (where the Ser522 phosphorylation site was abolished), led to improved motor function and prolonged survival in SOD1 G93A mice. Deletion of both copies of the Crmp1 gene ( Crmp1 −/− ) in SOD1 G93A mice resulted in deterioration of motor function . However, our case showed normal motor development, and further studies may be needed to identify the factors contributing to phenotypic heterogeneity. Section title: Discussion Educational score: 1.683632731437683 Domain: biomedical Document type: Other Language: en The P1 we report exhibited a broader range of phenotypes, and he was treated as follows: Section title: Discussion Educational score: 2.6952688694000244 Domain: biomedical Document type: Other Language: en Following the systematic treatment, the child demonstrated increased eye contact compared to before, improved completion of two- or three-steps, increased initiation of communication when needed, richer content in questions asked, more complete sentence structure compared to before, the ability to perceive the emotions of family members, attention to the movements of people around, and the capacity to make simple responses. There was also an improvement in puzzle-solving and computational skills, along with a slight improvement in attention. Section title: Conclusion Educational score: 3.8840739727020264 Domain: biomedical Document type: Study Language: en Currently, the CRMP1 gene has no clear disease phenotype association in the OMIM database. The CRMP1 (p.K586Rfs*75) frameshift variant was identified in this patient, with the following phenotypes: autism/autism spectrum disorder, stereotypic behavior, social impairment, delayed speech and language development, poor eye contact, and attention deficit hyperactivity disorder. Our report may provide evidence for an association between the CRMP1 gene and neurodevelopmental disorders. | Review | biomedical | en | 0.999998 |
PMC11695372 | Section title: Introduction Educational score: 3.9246339797973633 Domain: biomedical Document type: Review Language: en The novel coronavirus pneumonia (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been ravaging the world over the past 3 years. Although COVID-19 as a public health emergency of international concern (PHEIC) was declared to have ended by the WHO on 5 May 2023 ( 1 ), the pandemic itself is still far from over. As an RNA virus, SARS-CoV-2 mutates easily, leading to the emergence of numerous variants capable of evading the humoral immune system ( 2 , 3 ). In less than 4 years, the dominant strains have shifted from Alpha, Delta, to Omicron ( 4 , 5 ). The pace of vaccine development could not keep up with the speed of virus mutation. COVID-19 remains a significant threat to human society and more COVID-19 vaccine candidates should be developed. Section title: Introduction Educational score: 4.183794975280762 Domain: biomedical Document type: Study Language: en Recombinant subunit vaccines, with their advantage of large-scale production and transportation, have been widely used in the development of COVID-19 vaccines ( 6 ). Several COVID-19 subunit vaccines, such as ZF2001, are based on the receptor-binding domain (RBD) ( 7 ). While most of the antigenic regions are found in the RBD, the N-terminal domain (NTD) of the S protein is another crucial region for inducing neutralizing antibodies (nAbs) against COVID-19 ( 8 , 9 ). Several human nAbs that bind to the NTD, such as COV2-2676 and COV2-2489, had been developed and demonstrated both effective prophylactic and therapeutic efficacy against SARS-CoV-2 infection ( 9 – 11 ). Moreover, macaques immunized with a combination of NTD and RBD immunogens could be totally protected from lethal SARS-CoV-2 challenges ( 12 ), highlighting the promising potential of the NTD in vaccine development. Section title: Introduction Educational score: 4.151867866516113 Domain: biomedical Document type: Study Language: en Fc fusion proteins, which are composed of an immunoglobin Fc region and a desired linked protein, serve as an effective backbone for drug development ( 13 ). To date, 11 Fc fusion proteins have been approved by the Food and Drug Administration (FDA) ( 14 ). The Fc region could bind to the neonatal Fc receptor to prolong the plasma half-life and increase the immunogenicity of the Fc-fusion proteins ( 15 ). In our previous study, we developed RBD-Fc and RBD delta -Fc fusion vaccine candidates, both of which were able to induce humoral and cellular immune responses in mice ( 6 , 16 ). This study aims to develop a two-component recombinant vaccine using RBD-Fc and NTD-Fc, optimizing their ratio to achieve enhanced humoral and cellular immune responses. It is the first combination of RBD-Fc and NTD-Fc for COVID-19 vaccine development. This vaccine was also applied in a sequential immunization after two doses of inactivated vaccination. Humoral responses such as immunoglobulin G (IgG) and nAb were monitored for 27 weeks. The IFN-γ- and IL-4-producing cells, as well as the secretion of IL-10 and IL-2, were also measured. Through these experiments, we demonstrated that the two-component vaccine based on the RBD-Fc and NTD-Fc proteins exhibited strong immunogenicity and might represent a promising candidate for the COVID-19 vaccine development. Section title: Cells and viruses Educational score: 4.125795841217041 Domain: biomedical Document type: Study Language: en Vero E6 cells (NCACC) were cultured at 37℃ under 5% CO 2 in Minimal Essential Medium (Gibco, Waltham, MA, USA) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS). The prototype SARS-CoV-2 strain (12#) and the SARS-CoV-2 Omicron strain (BA.2) were obtained as mentioned previously ( 17 , 18 ). Viruses were propagated in Vero E6 cells, and the 50% tissue culture infective dose (TCID 50 ) was calculated by the Reed and Muench method ( 17 ). Section title: Mouse experiments Educational score: 4.083128452301025 Domain: biomedical Document type: Study Language: en In the two-dose immunization experiment, female BALB/c mice (8 weeks old, n = 5/group) were intramuscularly immunized with different ratios of RBD-Fc and NTD-Fc proteins (9:1, 3:1, 1:1, 8:0, 0:8, 1:3, and 1:9), with a total protein dose of 8 μg per mouse. Aluminum hydroxide was used as an adjuvant, mixed with immunized protein at a final solution of 0.5 mg/mL. Mice were immunized on day 0 and boosted on day 7, and phosphate buffer saline (PBS) containing 0.5 mg/mL of aluminum hydroxide was used as a negative control. The sera were collected as per the schedule shown in Figure 1A . Section title: Mouse experiments Educational score: 4.065967559814453 Domain: biomedical Document type: Study Language: en For the sequential immunization experiment, seven groups of female BALB/c mice (8 weeks old, n = 5/group) were immunized with two doses of the inactivated SARS-CoV-2 vaccine (prototype strain) on days 0 and 7. On day 28, mice were boosted with RBD-Fc (8 μg), NTD-Fc (8 μg), RBD-Fc/NTD-Fc (9:1, 8 μg), RBD-Fc/NTD-Fc (3:1, 8 μg), inactivated SARS-CoV-2 vaccine (prototype strain), inactivated SARS-CoV-2 vaccine (omicron strain), and PBS, individually. Mice receiving three doses of PBS were used as negative control. The sera and spleens were collected as per the schedule shown in Figure 1B . The RBD-Fc protein and the inactivated SARS-CoV-2 vaccine were prepared as previously described ( 6 , 19 ). NTD-Fc was purchased from Sino Biological Company. Section title: Enzyme-linked immunosorbent assay Educational score: 4.122889995574951 Domain: biomedical Document type: Study Language: en Briefly, enzyme-linked immunosorbent assay (ELISA) plates were coated with 50 ng of RBD or NTD or S1 protein per well overnight in carbonate-bicarbonate buffer (pH 9.6) and then blocked with 10% FBS in PBS. Serum samples were serially diluted twofold and added to each well. After three washes, the plates were incubated with rabbit anti-mouse IgG-HRP antibody at a dilution of 1:20,000 (Abcam, Cambridge, UK) for 1 h at 37℃. The color development reaction was performed using tetramethylbenzidine (TMB) and stopped with 2 M H 2 SO 4 . Absorbance was measured at 450 nm, and the endpoint dilution titer was defined as the highest reciprocal dilution of serum that produced an absorbance value at least 2.1 times above the background. Section title: Neutralization assay Educational score: 4.128098487854004 Domain: biomedical Document type: Study Language: en Mouse serum was inactivated at 56℃ for 30 min. Serial 2-fold dilutions of the sera were incubated with 100 TCID 50 of SARS-CoV-2 virus (prototype or omicron strain) for 1 h. The virus–serum mixtures were added to the Vero E6 cell-seeded 96-well plates and incubated for 3 days. Cytopathic effect (CPE) was observed in each well, and the neutralization titers were calculated as the reciprocal of the highest serum dilution than can protect 50% of wells from infection ( 16 ). Section title: Enzyme-linked immunospot assay Educational score: 4.094871997833252 Domain: biomedical Document type: Study Language: en The enzyme-linked immunospot assay (ELISPOT) assays were performed according to the instructions of IL-4 and IFN-γ ELISPOT kits. Twenty-seven weeks after vaccination, mice in the sequential immunization groups were euthanized, and their spleens were collected. Splenocytes were isolated by pressing spleens through the cell strainers and cultured in ELISPOT plates at a density of 2 × 10 5 per well for the IFN-γ and IL-4 detection. A cocktail containing RBD (1 μg/well) and NTD (1 μg/well) was used as the stimulant. Spot-forming cells (SFCs) were imaged with a ChemiDoc XRS+ imaging system (Bio-Rad, CA, USA) and analyzed using the Quantity One software. Section title: IL-2 and IL-10 detection Educational score: 4.0718488693237305 Domain: biomedical Document type: Study Language: en Splenocytes were prepared as described in the ELISPOT section and seeded in a 96-well plate at a density of 2 × 10 5 per well. After stimulation with RBD-NTD mixture (1:1, 2 μg/well) for 16 h, the supernatants of each well were obtained and the concentrations of secreted IL-2 and IL-10 were measured by ELISA kits (BD Biosciences, NJ, USA). Section title: Statistical analysis Educational score: 2.4476377964019775 Domain: biomedical Document type: Study Language: en All data were analyzed with GraphPad Prism 8.0. The Student’s t -test was used for comparisons between two groups, and p < 0.05 was considered as statistically significant. Section title: The immunogenicity of the two-component vaccines Educational score: 4.158207893371582 Domain: biomedical Document type: Study Language: en To determine the optimal RBD-Fc/NTD-Fc ratio for immunization, different RBD-Fc/NTD-Fc (R/N) ratios (9:1, 3:1, 1:1, 8:0, 0:8, 1:3, and 1:9) with a total protein amount of 8 μg for each mouse were tested. Mice were immunized and boosted after 1 week. Two weeks following the initial immunization, the IgG seroconversion rate was 100% in all groups . However, the geometric mean titer (GMT) of IgG antibodies in the R/N (0:8) group was 696, significantly lower than the other groups. Four weeks post-vaccination, IgG levels in all groups increased sharply. The GMTs of IgG antibodies in the R/N (9:1, 3:1, 1:1, 8:0, 0:8, and 1:3) groups were 89,144, 77,604, 38,802, 102,400, 2,786, and 33,779, significantly higher than those of each corresponding group at 2 weeks post-vaccination. In the R/N (1:9) group, the GMT of IgG antibodies was 19,401 at 4 weeks post-vaccination, rising to 25,600 2 weeks later, which was significantly higher than that at 2 weeks post-vaccination. The high IgG levels could last for at least 18 weeks. The GMTs of IgG antibodies in the R/N (9:1, 3:1, 1:1, 8:0, 0:8, 1:3, and 1:9) groups at 18 weeks post-vaccination were 102,400, 102,400, 51,200, 117,627, 3,200, 77,605 and 102,400, respectively. Section title: The immunogenicity of the two-component vaccines Educational score: 4.088810443878174 Domain: biomedical Document type: Study Language: en In addition to the RBD protein, S1 and NTD proteins were also coated onto the microwells of ELISA plates. As shown in Figure 2B , the GMTs of IgG antibodies in the R/N (9:1, 3:1, 1:1, 8:0, and 1:3) groups using S1-coated plates (left, blue background) were significantly higher than those using the NTD-coated plates (right, yellow background) at 2 weeks post-vaccination. Furthermore, the GMT of IgG antibodies in the R/N (0:8) groups using S1-coated plates was significantly lower than that using the NTD-coated plates at 2 weeks post-vaccination. Similar results were observed at 18 weeks post-vaccination. Using S1-coated plates, the GMTs of IgG antibodies in the R/N (9:1, 3:1, 1:1, 8:0, 0:8, 1:3, and 1:9) groups at 18 weeks post-vaccination were 77,605, 67,559, 29,407, 89,144, 4,850, 33,779, and 38,802, respectively. Among all groups, the GMTs of IgG antibodies in the R/N (9:1, 3:1, and 8:0) groups were relatively high when using both RBD- and S1-coated plates. Section title: The immunogenicity of the two-component vaccines Educational score: 4.125151634216309 Domain: biomedical Document type: Study Language: en To further evaluate the immunogenicity of the two-component vaccines, we measured the titers of nAbs using the prototype SARS-CoV-2 virus. The seroconversion rates of nAbs in the R/N (9:1, 3:1, 1:1, 8:0, 1:3, and 1:9) groups were 100% at 2 weeks post-vaccination, while that in the R/N (0:8) group was only 40% . Similar to the IgG titers, the nAb titers in all groups increased sharply at 4 weeks post-vaccination. By this time, the seroconversion rate of nAbs in the R/N (0:8) group had risen to 100%, and the GMTs of nAbs in R/N (9:1, 3:1, 1:1, 8:0, 1:3, and 1:9) groups were significantly higher than those of the corresponding groups at 2 weeks post-vaccination. The GMTs of nAbs in R/N (9:1, 3:1, 1:1, 8:0, 0:8, 1:3, and 1:9) groups were 127, 221, 222, 140, 16, 112, and 139, respectively, with the R/N (3:1, 1:1) groups showing the highest levels. At 18 weeks post-vaccination, the GMTs of nAbs in the R/N (9:1, 3:1, 1:1, 8:0, 0:8, 1:3, and 1:9) groups were 354, 299, 269, 231, 17, 232, and 267, respectively, with the R/N (9:1, 3:1) groups exhibiting slightly higher titers compared to the others. Considering the GMTs of IgG antibodies and nAbs at 18 weeks post-vaccination, we selected the RBD-Fc/NTD-Fc ratios of 9:1 and 3:1 for the two-component vaccines. Section title: The humoral immune response in the sequential immunization Educational score: 4.137515068054199 Domain: biomedical Document type: Study Language: en To verify the effectiveness of the two-component vaccines in the heterologous immunization, mice were first immunized with two doses of prototypic inactivated SARS-CoV-2 vaccines on day 0 and day 7. Subsequently, the mice received a booster with RBD-Fc (8 μg), NTD-Fc (8 μg), R/N (9:1, 8 μg), R/N (3:1, 8 μg), the prototypic inactivated SARS-CoV-2 vaccine, the inactivated omicron vaccine, or PBS on day 28. Five weeks post-vaccination (1 week after the third immunization), the GMTs of IgG antibodies in the RBD, NTD, R/N (9:1, 3:1), prototype, and omicron groups were all significantly higher than those at 3 weeks post-vaccination (1 week after the second immunization). The elevated levels of IgG antibodies persisted for at least 22 weeks, with GMTs of 89,144, 51,200, 102,400, 117,626, 77,605, and 77,605 for the RBD, NTD, R/N (9:1), R/N (3:1), prototype, and omicron groups, respectively . Using S1-coated ELISA plates (left, blue background), the GMTs of IgG antibodies in the RBD, NTD, R/N (9:1, 3:1), prototype, and omicron groups were also high, similar to the results obtained using RBD-coated plates . However, compared with the results from NTD-coated plates (right, yellow background), the GMTs of IgG antibodies in all groups using S1-coated plates were higher at both 3 and 27 weeks post-vaccination. Section title: The humoral immune response in the sequential immunization Educational score: 4.112531661987305 Domain: biomedical Document type: Study Language: en Similar to the IgG antibody titers, the nAbs against the prototype SARS-CoV-2 virus were also increased significantly at 1 week after the third vaccination. The GMTs of nAbs in the RBD, NTD, R/N (9:1, 3:1), prototype, and omicron groups were 139, 42, 185, 255, 323, and 134, respectively. The GMTs of nAbs continued to rise over time, peaking at 15 weeks post-vaccination. These elevated nAb titers could sustain for at least 22 weeks. At 27 weeks post-vaccination, the GMTs of nAbs in the RBD, NTD, R/N (9:1), R/N (3:1), prototype, omicron, and PBS groups were 806, 806, 1,333, 935, 639, 708, and 255, respectively, still significantly higher than those at 3 weeks post-vaccination. Based on the results of IgG and nAbs, both the RBD-Fc/NTD-Fc ratios of 9:1 and 3:1 appeared to be suitable for the two-component vaccine. Section title: The neutralizing antibody against the SARS-CoV-2 omicron variant Educational score: 4.108339786529541 Domain: biomedical Document type: Study Language: en The omicron variant (BA.2) was also utilized to assess the nAb levels in both the two-dose immunization and the sequential immunization. In the two-dose immunization, the GMTs of nAbs at 18 weeks post-vaccination in the R/N (9:1, 3:1, 1:1, 8:0, 1:3, and 1:9) groups were 14, 40, 13, 24, 3, and 8 . Notably, nAbs were undetectable in the R/N (0:8) group. The GMTs of nAbs in R/N (3:1 and 8:0) groups were both significantly higher than those in the R/N (1:3) group. In the sequential immunization, the GMTs of nAbs at 27 weeks post-vaccination in the RBD, NTD, R/N (9:1), R/N (3:1), prototype, omicron, and PBS groups were 42, 9, 27, 44, 10, 46, and 9, respectively . The GMTs of nAbs in RBD, R/N (3:1), and omicron groups were significantly higher than those in R/N (1:3) groups. Overall, these results suggested that the R/N (3:1) group might induce a relatively high level of nAbs compared to the other groups, making it a potential candidate for the two-component vaccine. Section title: The cellular immune response in the sequential immunization Educational score: 4.085790157318115 Domain: biomedical Document type: Study Language: en Next, we evaluated the cellular immune response of the two-component vaccines in the sequential immunization using ELISPOT assay. Compared to the PBS group, a significantly higher number of IFN-γ SFCs in the RBD, NTD, R/N (9:1), R/N (3:1), prototype, and omicron groups were found, with counts of 120, 131, 113, 159, 124, and 117, respectively . Similar results were found from the IL-4 ELISPOT assay, where the number of IL-4 SFCs was greater in the RBD, NTD, R/N (9:1), R/N (3:1), prototype, and omicron groups compared to the PBS group . Section title: The cellular immune response in the sequential immunization Educational score: 4.0818376541137695 Domain: biomedical Document type: Study Language: en We also measured the levels of IL-2 and IL-10 secreted by the splenocytes of immunized mice using ELISA. As shown in Figures 5C, D , the concentrations of IL-2 and IL-10 in the three dose groups such as RBD, NTD, R/N (9:1), R/N (3:1), prototype, and omicron groups were higher than those in the two dose PBS group. The IL-10 concentrations of the RBD, NTD, R/N (9:1), R/N (3:1), prototype, and omicron groups were 181.2, 105.3, 80.3, 105.3, 100.8, and 95.8, respectively. The IL-2 concentrations of those groups were 219.7, 257.3, 193.1, 203.2, 173.5, and 241.1. Section title: The cellular immune response in the sequential immunization Educational score: 2.580705404281616 Domain: biomedical Document type: Study Language: en All results showed that a third vaccination could effectively stimulate a robust cellular immune response. Section title: Discussion Educational score: 4.066601753234863 Domain: biomedical Document type: Study Language: en While COVID-19 is no longer classified as a public health emergency, it is still a significant challenge to the whole society. Here, we have first developed a two-component recombinant vaccine for COVID-19 based on RBD-Fc and NTD-Fc proteins, with an optimal RBD-Fc/NTD-Fc ratio of 3:1. This two-component vaccine is capable of inducing durable and potent IgG and nAbs in both the homologous and heterologous immunization experiments. Additionally, it effectively triggers robust cellular immune response. Overall, this two-component vaccine exhibits great potential as a COVID-19 vaccine candidate. Section title: Discussion Educational score: 4.359963417053223 Domain: biomedical Document type: Study Language: en The design of immunogens is critical for the vaccine development. Both RBD and NTD were located at the S1 subunit of spike (S) protein, which binds to the host cell receptor, angiotensin-converting enzyme 2 (ACE2), facilitating viral entry into cell ( 20 ). The RBD, as the receptor binding site for the ACE2 receptor, is an ideal immunogen, and over 11 COVID-19 vaccines, including ZF2001 (Zhifei) and CoVaccine (WestVac), have been developed based on the RBD protein ( 21 , 22 ). In contrast, the NTD has been less extensively investigated and used for vaccine development. Cumulative evidence indicates that lots of neutralizing epitopes are present in the NTD. nAbs such as 4A8, have been identified, along with specific epitope sites such as D144‐Q158 and E246‐T253 within the NTD ( 23 , 24 ). Analysis of memory B cells from SARS-CoV-2-infected individuals had shown that anti-NTD antibodies could neutralize SARS-CoV-2 variants including Omicron ( 25 ). Additionally, the NTD plays a crucial role in host receptor recognition. Díaz-Salinas ( 26 ) reported that the NTD bound terminal sialic acid to enhance both SARS-CoV-2 S-mediated virus attachment and ACE2 binding. Mouse hepatitis coronavirus, a betacoronavirus, utilizes the NTD to recognize its host receptor, CEACAM1a ( 27 ). These findings suggested that the NTD might be another valuable immunogen alongside the RBD. Inclusion of NTD could enhance the immunogenicity of the RBD subunit vaccine, and fusing the NTD onto the RBD–RBD protein enhances T-cell immunity, which, in turn, supports B-cell immunity and overall improves the immunogenicity of the antigen ( 28 ). A cocktail of NTD-directed and RBD-targeting nAbs could also work synergistically to provide protection against SARS-CoV-2 ( 12 ). In the two-dose immunization assay, the GMTs of nAbs in the two-component vaccine groups (R/N = 9:1, 3:1, and 1:1) were 354, 299, and 269 at 18 weeks post-vaccination, slightly higher than those in the single-component vaccine group (RBD-Fc, R/N = 8:0; NTD-Fc, R/N = 0:8). Similar trends were also found in the heterologous immunization. The GMTs of nAbs in the two-component vaccine groups (R/N = 9:1, 3:1) were 1,333 and 935, compared to those of 806 and 805 in the RBD-Fc and NTD-Fc vaccine groups. When using the omicron strain in the neutralization assay, the GMT of nAbs in the R/N = 3:1 group was 40, which was still slightly higher than that of 24 in the RBD-Fc vaccine group. These results indicated that the two-component vaccine, consisting of RBD-Fc and NTD-Fc immunogens, exhibited robust immunogenicity than the single component vaccine at the same dose. The combination of NTD-Fc and RBD-Fc might have a synergistic effect on the immune system. Furthermore, when NTD-Fc was used as a single component vaccine, the GMT of nAbs in the two-dose immunization was only 17 at 18 weeks post-vaccination, significantly lower than those in the two-component vaccine groups and the RBD-Fc vaccine group. Although NTD contains several neutralizing epitopes, it may not be advisable to use NTD-Fc as the sole immunogen. Section title: Discussion Educational score: 4.090041160583496 Domain: biomedical Document type: Study Language: en Since the S1 subunit comprises both the RBD and NTD, it is normal to question why we do not use S1 instead of RBD and NTD for vaccine development. However, in our previous study, we found that the S1-Fc protein exhibited poorer immunogenicity compared with the RBD-Fc group ( 6 ). Similar results were also found when comparing the S1 protein with the RBD protein ( 29 ). One reason for this might be that the two distinct proteins expose more key epitopes than a single combined protein, as some epitopes could be obscured by conformation changes. Although RBD and NTD were both located at the S1 subunit, S1 might not be an ideal immunogen for vaccine development. Section title: Discussion Educational score: 4.214720249176025 Domain: biomedical Document type: Study Language: en As a two-component vaccine, the ratio of the RBD-Fc and NTD-Fc is a key parameter. The combined RBD and NTD immunogens with the ratio of 1:1 exhibited more robust immunogenicity than a single RBD or NTD immunogen at the same dose ( 12 ). Furthermore, the nAbs induced by RBD were generally more potent than those induced by NTD ( 25 ). The 1:1 ratio of RBD to NTD might not be optimal for immunization. Therefore, we tested additional ratios, including 9:1, 3:1, 1:3, and 1:9. In the two-dose immunization, the GMTs of IgG antibodies and nAbs in the R/N (9:1 and 3:1) groups were slightly higher than the R/N (1:1, 1:3, and 1:9) groups. In the heterologous immunization using the omicron variant for detection, the GMT of nAbs in the R/N (3:1) group was 44, compared to 27 in the R/N (9:1) group. Among all tested ratios, the RBD-Fc/NTD-Fc 3:1 group exhibited the highest sustained nAb titers, suggesting its optimal immunogenicity, while the RBD monomer has low immunogenicity, and the RBD dimer could enhance the immunogenicity ( 7 , 30 ). By conjugating the human IgG Fc fragment to the C-terminus of the RBD protein, we previously constructed an RBD-Fc fusion protein, which exhibited superior immunogenicity in mice than the RBD protein. Fc might promote vaccine uptake by antigen-presenting cells (APCs), boosting the RBD- or NTD-stimulated immune capacity ( 16 ). Section title: Discussion Educational score: 4.086668491363525 Domain: biomedical Document type: Study Language: en In this study, we first introduced NTD-Fc combined with RBD-Fc as a two-component vaccine. This combination was able to induce potent and durable nAbs against SARS-CoV-2, as well as a robust cellular immune response. Moreover, no significant damage was detected in the lung slices of the immunized mice , indicating the safety of this two-component vaccine. Section title: Discussion Educational score: 4.109044551849365 Domain: biomedical Document type: Study Language: en Though COVID-19 has become endemic, the SARS-CoV-2 virus continues to evolve to adapt the herd immunity ( 31 ). As of the summer of 2024, COVID-19 cases surged worldwide, with subvariants such as JN.1 and KP.3 displaying significant immune evasion ( 32 ). Nevertheless, vaccine is still one of the most effective measures to combat COVID-19. Given that most individuals in China have received at least two vaccinations, heterologous booster seems to be a promising vaccination strategy ( 33 ). In our study on heterologous immunization , the two-component vaccine (R/N = 3:1) and the RBD-Fc vaccine induced significantly higher GMT of nAbs than that of the prototype inactivated vaccine (homologous group) when using the omicron strain. The GMTs of nAbs in the third immunization groups (RBD, NTD, R/N = 9:1, R/N = 3:1, prototype, and omicron) were significantly higher than those in the PBS group that received only two doses of the inactivated vaccine . This indicated that an additional booster, particularly a heterologous booster, is beneficial. Section title: Discussion Educational score: 4.099031448364258 Domain: biomedical Document type: Study Language: en Cellular immune response is pivotal for the vaccine efficacy, providing long-term protection for COVID-19 ( 34 ). In our study, results demonstrated that a second booster could trigger a significantly cellular immune response in both the homologous and heterologous immunization . The secretion of IFN-γ and IL-2 produced by Th1-cells, as well as IL-4 and IL-10 produced by Th-2 cells, was found to be increased. It seemed that the two-component vaccine induced a relatively balanced Th1- and Th2-cell immune responses. Section title: Discussion Educational score: 4.07724142074585 Domain: biomedical Document type: Study Language: en In summary, our study is the first to employ the RBD-Fc and NTD-Fc as a two-component vaccine. This formulation demonstrated the ability to elicit high levels of IgG and nAbs, which could last for more than 6 months. The vaccine also induces a robust cellular immune response, and the optimal ratio of RBD-Fc to NTD-Fc was identified as 3:1. As the need for diverse COVID-19 vaccines remains critical in anticipation of future outbreaks, this two-component vaccine might be a promising candidate for further COVID-19 vaccine development. | Review | biomedical | en | 0.999997 |
PMC11695373 | Section title: Introduction Educational score: 4.3539958000183105 Domain: biomedical Document type: Review Language: en Pyramidal neurons (PNs) receive and integrate 1,000's of synaptic inputs impinging onto their dendritic arbor to shape the neuronal output. The richness and the complexity of such input-output transformation primarily relies on the ability of neurons to generate different forms of dendritic local spikes, regenerative events originating in the dendrites profoundly influencing the probability and the temporal structure of somatic spiking. Extensive work during the last two decades has identified the impact of clustering and cooperative plasticity among glutamatergic synapse in promoting dendritic spikes. However, the role of inhibitory synapses in such processes remains elusive. In this opinion paper, following a general introduction on the impact of the synaptic input spatial distribution in neuronal activity, we highlight the coordinated plasticity of excitatory and inhibitory dendritic synapses as an emerging key factor in the organization of the dendritic input architecture. In particular we will emphasize that the relative positioning of diverse excitatory and inhibitory dendritic synapses at the microscale level is a major determinant for shaping dendritic dynamics and neuronal circuit function in the brain. Section title: Multiscale spatial arrangement of dendritic excitatory or inhibitory synaptic inputs Educational score: 4.705974102020264 Domain: biomedical Document type: Study Language: en In different brain areas, distinct synaptic inputs converging onto PNs show a macro-scale distribution across large dendritic compartments. For instance, in the hippocampal formation, excitatory fibers from entorhinal cortex (EC) project to the distal portions of apical dendrites of CA1 PNs through the perforant path (PP), while Schaffer collaterals (SCs) from the CA3 area mainly contact the proximal dendrites . Similarly, in the neocortex, intra-cortical layer 2/3 (L2/3) PNs axons (feedforward information) contact the proximal dendrites of layer 5 (L5) PNs, with cortico-cortical inputs from high-order cortical areas (feedback information) targeting their distal dendrites . This illustrates a large-scale connectivity scheme wherein fibers from either distant or local brain regions preferentially contact distal or proximal dendrites, respectively . Such broad-scale input organization reflects important functional properties where the activation of proximal dendrites typically produces single action potentials while co-activation of distal and proximal synaptic inputs can generate calcium plateau potentials—specific forms of dendritic spikes initiated in the distal dendritic region—leading the neuron to burst firing . This supra-linear integration provides the biophysical basis for a fundamental associative process to combine and compare different types of information at the single cell level . Along the same line, the differential effect of distal feedforward inputs triggering single spikes and the combined activation of feedforward and distal feedback inputs inducing burst firing, provides the opportunity for the independent transmission of these two distinct signals through the same neuronal pathway . Section title: Multiscale spatial arrangement of dendritic excitatory or inhibitory synaptic inputs Educational score: 4.458261013031006 Domain: biomedical Document type: Study Language: en Intriguingly, GABAergic inputs are also non-randomly distributed along the axo-dendritic axis of PNs. Diverse subclasses of GABAergic interneurons (INs) target specific sub-regions of PNs including axon initial segment, soma, proximal dendrites and distal dendrites, with a specific temporal activation critically contributing to e.g., brain oscillations . In both hippocampus and neocortex, the proximo-distal dendritic compartmentalization of diverse GABAergic inputs creates a spatial pattern where distinct GABAergic fibers broadly align with specific subsets of excitatory inputs. For example, in the hippocampus, oriens-lacunosum-moleculare (O-LM), neurogliaform, and perforant path (PP)-associated INs target the distal dendrites of CA1 pyramidal neurons aligning with PP inputs from the EC. Comparably, bistratified, SC-associated and Ivy interneurons match glutamatergic inputs from CA3 onto proximal dendrites . Section title: Multiscale spatial arrangement of dendritic excitatory or inhibitory synaptic inputs Educational score: 4.856154918670654 Domain: biomedical Document type: Study Language: en The existence of structured patterns of synaptic inputs localization persists at smaller scales. At glutamatergic side, computational and experimental works showed that dendritic synaptic inputs clustering favors dendritic spikes initiation . In L5 PNs, for instance the activation of glutamatergic inputs within a ~ 40 μm range undergo supra-linear summation due to N-methyl-D-aspartate (NMDA) receptor-dependent regenerative mechanism, whereas inputs more than 80 μm apart integrate linearly, indicating the key role of the spatial determinants in dendritic input summation . The functional clustering of glutamatergic inputs has been observed directly in dendrites of both CA3 and L2/3 PNs, where spontaneous activity is more likely to co-activate neighboring glutamatergic spines rather than distant spines, thus forming glutamatergic synaptic “assemblets” within ~ 10 μm . The clustered organization of glutamatergic inputs underpins an important role at the functional level. In the visual cortex, the clustering of similarly tuned inputs aids edge detection and contour integration , while in the motor cortex, task-related inputs cluster within 10 μm subdomains to support decision-making . Besides the relevance of the tight spatial proximity between active glutamatergic synapses ( synaptic clustering ), the initiation of dendritic spikes strongly depends on the dendritic morphology. In thin and short dendritic branches, the high input resistance determines low attenuation of the depolarization produced by individual synapses thus promoting the signal summation within the branch . For instance, the timely activation of ~ 20 glutamatergic inputs on a radial oblique dendritic branch of 100 μm in CA1 PNs initiate a local sodium spike regardless of their spatial relationship along the branch, thus determining in-branch clustering . Anatomical studies of SCs synapses localization onto CA1 PNs dendrites have revealed a highly non-uniform connectivity structure. In particular, the number of short inter-spine distances as well as the number of glutamatergic inputs per branch was greater than chance level, thus supporting both synaptic clustering and in-branch clustering modes, respectively . Similar findings were observed for thalamocortical inputs onto L5 PN . Collectively, this evidence indicates that, at different scales, the spatial arrangement of glutamatergic synapses in dendrites of PNs crucially shapes the transfer function between synaptic activation and dendritic depolarization/spiking . It is interesting to note that, synaptic inputs in dendrites of interneurons are less spatially structured with respect to PNs , and, in contrast to PNs, synaptic inputs in small caliber dendrites of fast spiking basket cells tend to summate sub-linearly . Section title: Multiscale spatial arrangement of dendritic excitatory or inhibitory synaptic inputs Educational score: 4.6354169845581055 Domain: biomedical Document type: Study Language: en As with excitatory inputs, several lines of evidence show that synaptic inhibition in PNs dendrites depends on local spatial determinants at the microscale level, such as their fine relative positioning with respect to excitatory synapses . Modeling studies suggest that GABAergic synapses positioned distally (off-path) from a cluster of glutamatergic synapses more efficiently raise the threshold for initiating a dendritic spike compared to proximally-placed ones (on-path), whereas the on-path location is more effective in shunting already-triggered dendritic spikes . Both predictions have been corroborated experimentally ex vivo in L5 PNs, confirming that the specific spatial arrangement of GABAergic synapses in dendritic branches is an important determinant shaping dendritic excitability . In this concern, studies report that diverse GABAergic inputs from specific interneurons are highly structured at branch and sub-branch levels. In CA1 PNs, O-LM interneurons (somatostatin+, SOM+) or neurogliaform interneurons (neural nitric oxide synthase+, nNOS+) preferentially target the ending or the intermediated region of the terminal domain of distal dendrites, respectively whereas bistratified interneurons (neuropeptide Y+, NPY+) target the origin of the terminal domain of proximal apical oblique and basal dendrites . In addition, the study of the excitatory and inhibitory synapses distribution in the whole dendritic arbor in L2/3 PNs revealed that, while density of both synapses significantly vary in different neuronal sub-regions, its ratio was remarkably balanced at branch level . Finally, inhibitory GABAergic synapses can be located directly on glutamatergic spines thus effectively controlling spine depolarization . Section title: How does cooperative plasticity among glutamatergic synapses shape synaptic clustering? Educational score: 4.841662406921387 Domain: biomedical Document type: Study Language: en Extensive work on glutamatergic spines reports that the expression of long-term potentiation (LTP) at an individual spine can lower the threshold for the induction of synaptic plasticity at neighboring spines by spreading signaling molecules such as small GTPases from the potentiated spine in dendritic stretches of ~10 μm: this establishes the coordinated potentiation of a subset of contiguous spines ultimately leading to the formation of a glutamatergic synaptic cluster . On the other hand, the stimulation of a glutamatergic spine cluster can depress nearby spines through the diffusion of the phosphatase calcineurin, a mechanism that is expected to increase the structural and functional identity of specific clusters . Likewise, long-term depression (LTD) at an individual spine can either depress or potentiate neighboring spines . Overall, these observations suggest that short-range interplay between spines can define the spatial pattern of dendritic glutamatergic synapses. Nevertheless, how GABAergic synapses contribute to these processes remains largely obscure. Traditionally, inhibition has been considered poorly plastic and to take part to plasticity phenomena mainly by adjusting the threshold for the induction of glutamatergic plasticity . In this concern, modeling studies report that specific placement of GABAergic synapses with respect to either excitatory synapses or dendritic branches can spatially constraint glutamatergic plasticity hence influencing the degree of glutamatergic synapses clustering . Similarly, the activation of GABAA receptors by GABA uncaging leads to the shrinkage of nearby glutamatergic spines within a range of ~15 μm, reinforcing the spatial role of inhibition in promoting the competitive selection of dendritic spines . Section title: How does cooperative plasticity among glutamatergic synapses shape synaptic clustering? Educational score: 4.595800399780273 Domain: biomedical Document type: Study Language: en Nevertheless, several lines of evidence indicate that GABAergic synapses express several forms of plasticity . This prompts the questions of how glutamatergic and GABAergic plasticity interact at dendritic level at the microscale level and how this can shape synaptic clustering—topics that have thus far been investigated mainly through indirect approaches . After the induction of spike-timing-dependent plasticity at a specific synaptic population subset in an auditory cortex PN, the plasticity of excitatory and inhibitory plasticity at distinct unstimulated synaptic population subset was found to be co-tuned to achieve a precise excitation-to-inhibition set point . Interestingly, the plasticity-induced remodeling of excitatory and inhibitory synapses on dendrites of L2/3 PNs in the visual cortex is spatially coordinated in dendritic portions of ~10 μm suggesting short-range interplay between inhibitory and excitatory synapses . In addition, the stimulation of thalamic afferents to distal dendrites of cortical L2/3 PNs induces inhibitory LTP at GABAergic synapses formed by SOM+ interneurons in the same dendritic portion, thus hinting to local interaction between excitatory and inhibitory synapses . Extending this framework, a modeling study identifies the presence of plastic GABAergic synapses as important organizers of dendritic glutamatergic synaptic clustering . Section title: How does cooperative plasticity among glutamatergic synapses shape synaptic clustering? Educational score: 4.704010963439941 Domain: biomedical Document type: Study Language: en A more recent work investigated the spatial determinants for the interaction between individual dendritic glutamatergic and GABAergic synapses in hippocampal neurons . By inducing single-spine LTP through the pairing of glutamate uncaging with somatic action potential train, they observed that GABAergic synapses located within a spatial range of ~3–4 μm around the potentiated spine were depressed. Although several factors could limit the generalization of this finding including the poorly physiological induction of LTP and the lack of in vivo data, the spatial dependence of the interaction between excitation and inhibition likely plays an important role in the organization of dendritic synaptic inputs. First, by considering the local effect of inhibition , this heterosynaptic interplay is expected to disinhibit specific potentiated glutamatergic inputs through a winner-takes-all process, with e.g., other concurrent plasticity phenomena maintaining the global dendritic homeostatic balance. Second, the activity-dependent depression of a neighboring GABAergic synapse can contribute to the formation of glutamatergic synaptic clusters thus complementing the cooperative plasticity phenomena between glutamatergic inputs mentioned above. Finally, in the light of this short-range interplay, the convergence of diverse excitatory and inhibitory inputs within the same dendritic stretch can crucially impact at the network level, allowing, for instance, specific glutamatergic inputs to differentially control inputs from different interneuron subtypes. For example, PP and thalamic inputs contact the distal apical dendrites of CA1 PNs together with inputs from O-LM and PP-associated interneurons, which primarily mediate feed-back and feed-forward inhibition, respectively. If, differently from thalamic inputs, EC inputs are consistently located within the “interplay range” with inputs from O-LM interneurons, EC activity could weaken neighboring O-LM inputs . This could bias the balance of inhibition from feedback to feed-forward, thereby altering how these dendrites process and integrate incoming signals. Thus, in analogy with the aforementioned large-scale matching between excitation and inhibition in proximal and distal dendritic compartments, it is important to define the co-alignment between excitatory and inhibitory inputs at the microscale level. The spatial pattern of diverse excitatory and inhibitory inputs along the dendrites may serve as a “fingerprint” for PN subtypes, where the consistent pairing of particular excitatory inputs with inhibitory inputs from specific interneurons could act as structural “synaptic motifs.” In a broader framework, the impact of excitatory-inhibitory short-range synaptic interplay can be assessed by including specific synaptic topology and plasticity rules in available biophysical computational models predicting the spiking output of PNs receiving realistic excitatory and inhibitory temporal activity patterns at cellular level. This will allow to understand how short-range plasticity contribute to modulate specific network oscillations by tuning at dendritic level the contribution of diverse interneuron subtypes, or how it could enable associative learning by differentially gating information from distinct brain areas. Importantly, this could also clarify how aberrant short-range plasticity could lead to the disruption of coordination between different interneurons subtypes activity ultimately causing pathology. In the long run, the refined information about the dendritic synaptic spatial arrangement and short-range interaction could be integrated in computational models that include dendritic computation in large-networks functions and will also contribute designing more neuromorphic and efficient deep neuronal networks . | Review | biomedical | en | 0.999996 |
PMC11695375 | Section title: Introduction Educational score: 4.199042320251465 Domain: biomedical Document type: Study Language: en Histiocytic sarcoma (HS) is a rare and aggressive lymphohematopoietic tumor derived from non-Langerhans histiocytic cells of the mononuclear macrophage system ( 1 , 2 ), which most often involves the lymph nodes and/or gastrointestinal tract ( 3 ). Primary central nervous system HS (PCNSHS) is extremely rare, accounting for less than 1% of all lymphohematopoietic neoplasms ( 4 ). Although the pathogenesis of HS remains unclear, histologically, PCNSHS is characterized by the infiltration of inflammatory cells in the central nervous system and plays a crucial role in confirming the diagnosis; CD68, CD163, and lysozyme are recognized as typical markers that distinguish PCNSHS from other hematopoietic neoplasms, such as B-cell or T-cell non-Hodgkin’s lymphoma ( 1 , 5 – 7 ). Unfortunately, there are no standard or effective treatment strategies for PCNSHS. Section title: Introduction Educational score: 3.672663927078247 Domain: biomedical Document type: Clinical case Language: en Here, we describe a patient with primary cerebellar HS and review the available literature on this aggressive disease. Section title: Clinical presentation Educational score: 3.9050137996673584 Domain: clinical Document type: Clinical case Language: en The treatment timeline is shown in Figure 1 . A 30-year-old woman presented with dizziness and headache that had persisted for three months. On February 23, 2023, magnetic resonance imaging (MRI) revealed lamellar abnormal signals at the right cerebellar hemispheres, approximately 3.6 cm*3.0 cm in size, with clear boundaries, slightly low signal on T1WI, equal or slightly high signal on T2WI, low signal envelope on the edge, and unrestricted diffusion. The lesions showed obvious uniform enhancement on the enhanced scan. Abnormal signals with slightly longer T1 and T2 images were observed around the lesions. The adjacent fourth ventricle and brainstem were compressed and narrowed, respectively. The ventricular system was slightly enlarged, and there were a few symmetrical distributions of slightly longer abnormal T1 and T2 signals around the bilateral ventricles . Preliminary diagnoses were medulloblastoma, pilocytic astrocytoma, or lymphoma. Section title: Treatment and pathology Educational score: 4.065066337585449 Domain: biomedical Document type: Clinical case Language: en On February 28, 2023, the patient underwent surgical debulking. Histologically, large cells with abundant cytoplasm were mostly present in cerebellar biopsies. Morphologically, these cells had characteristics of histiocytes, and immunohistochemistry showed GFAP (-), Oligo2 (-), P53 (+), ATRX (+, expressed), HMB45 (-), CK (-), H3K27ME3 (+, expressed), H3K27M (-), CD68 (PGM1) (+), CD163 (+), CD1a (-), langerin (-), and ALK (-). FISH test: No BRAF translocations were detected. Mutations in TERT (P250/P228), BRAF (V600E), or H3F3A/HIS1H3B (K27/G34/K36) were not detected. The examination revealed no clear glial differentiation . Based on the histopathological morphology and immunophenotype, the lesion was considered to be HS. Section title: Treatment and pathology Educational score: 3.9090707302093506 Domain: clinical Document type: Clinical case Language: en On April 25, 2023, two months after surgery, brain MRI showed falciform long T1 and T2 signal shadows was in the right cerebellum, FLAIR represented circular high signal, and annular enhancement was observed on the contrast scan. For staging evaluation, chest and abdominal Computed Tomography (CT), positron emission tomography (PET), and bone marrow examinations were performed . Only the brain lesions were found. To decrease the risk of recurrence, the patient underwent adjuvant radiotherapy at a dose of 60 Gy in 30 fractions. CT with a fixed mask was performed with the patient in the supine position. The RayStation treatment planning system (RaySearch Laboratories, USA) was used for radiation therapy, contouring, and planning. The daily dose of 2.0 Gy was delivered to the patient with an Elekta Linear Accelerator (Edge™) using intensity-modulated radiation therapy plus volumetric modulated arc therapy . The patients were evaluated weekly for radiation toxicity. After adjuvant RT was completed, head MRI was obtained every three months, and no evidence of residual neoplasm or tumor recurrence was found after RT. The patient remains in complete remission . Section title: Discussion Educational score: 3.47348952293396 Domain: biomedical Document type: Review Language: en According to the literature review, PCNSHS is a rare subtype of HS, and its occurrence in the central nervous system distinguishes it from HSs that may occur in other organs. PCNSHS often presents with non-specific symptoms, including headache, seizures, focal neurological deficits, and changes in mental status. Section title: Discussion Educational score: 4.068962097167969 Domain: biomedical Document type: Study Language: en Neurological HS images showed mainly brain parenchymal involvement with single lesions (60%). Commonly, CT is the first choice for HS lesions, which mostly represent high-density lesions due to the high nuclear-cytoplasmic ratio of tumor cells ( 8 ); however, MRI is better than CT. According to the MRI scan, the lesions are mostly round or oval, with clear boundaries, and are often located at the junction of the gray and white matter, which may be accompanied by cystic components, necrotic areas (20%), or hemorrhages (4%). This characteristic is helpful for identifying other diseases; however, it still lacks typical characteristics. The lesions mostly appear as T1 equal or slightly high signal, T2 low or slightly higher signal. PET examination revealed hypermetabolic changes ( 9 ). Section title: Discussion Educational score: 4.102965354919434 Domain: biomedical Document type: Study Language: en Histologically, PCNSHS is characterized by the infiltration of large pleomorphic histiocytes into the central nervous system ( 1 ). Immunohistochemistry plays a crucial role in confirming the diagnosis, with markers, such as CD68, CD163, and lysozyme, being commonly positive, and not expressing specific T or B cell markers, myeloid markers, follicular dendritic cell markers, or epithelial cell markers ( 1 , 8 , 10 ) ( Table 1 ). Section title: Discussion Educational score: 4.095211029052734 Domain: biomedical Document type: Study Language: en Unfortunately, there is no standard therapy for HS or PHSCNS. Patients were treated using multimodal strategies, such as surgery, chemotherapy, and/or radiation therapy. In contrast, surgical resection combined with adjuvant radiotherapy is recommended for patients with single lesions ( 7 ). Patients with multiple lesions have a more aggressive clinical course, and combined chemotherapy is recommended; however, the optimal chemotherapy regimen is unclear, and lymphoma treatment regimens are commonly used, such as the CHOP regimen (cyclophosphamide, doxorubicin, vincristine, and prednisone) alone or combined with Etoposide ( 10 ). Additionally, genomic sequencing may play an important role in the identification of molecularly targeted agents and immune checkpoint inhibitors. IDBAIH et al. reported a case of neurological HS with BRAF V600E mutation, and the BRAF inhibitor Vemurafenib was used to achieve a good therapeutic effect in the short term ( 11 ), and May et al. reported the use of Dasatinib in a case of PHSCNS with platelet-derived growth factor receptor mutation ( 4 ). However, even if PHSCNS received treatment, the median survival time is 7.0 ± 0.98 months (95% confidence interval: 5.08–8.92) and an average survival time is 24.07 ± 5.1 months (95% confidence interval: 14.08–34.06) ( Table 2 ) ( 2 , 12 – 15 ). Section title: Discussion Educational score: 3.242918014526367 Domain: biomedical Document type: Clinical case Language: en In this case, the disease was located in the cerebellum, and no metastasis was observed on PET. Thus, only radiation at a dose of 60 Gy was administered after surgery. To date, no recurrence has been reported, and the survival time was 23 months. Meanwhile, such integrated therapies with surgery and radiotherapy might be useful in all cases of sarcoma ( 16 , 17 ). Section title: Conclusion Educational score: 2.8855385780334473 Domain: biomedical Document type: Clinical case Language: en HS is an extremely rare disease with a poor prognosis, and intensive radiation-based chemotherapy is a treatment option. This case demonstrates that patients can benefit from radiotherapy for localized lesions. Furthermore, owing to its rarity, ongoing research aims to better understand the biology of HS and develop more effective treatment strategies. | Study | biomedical | en | 0.999997 |
PMC11695380 | Section title: 1 Introduction Educational score: 4.382116794586182 Domain: biomedical Document type: Review Language: en Lung diseases account for over four million premature deaths annually, and their prevalence is expected to rise in the coming years. Chronic respiratory diseases, including lung cancer, asthma, and chronic obstructive pulmonary disease (COPD), together represent the third leading cause of death worldwide . Repair of severe acute lung injury remains extremely challenging due to the lung’s limited ability to regenerate and the disordered environment formed by edema, inflammation, and fibrosis following lung disruption . Steady cell turnover helps the mature mammalian lung maintain homeostasis, and diverse cell populations are necessary for lung remodeling and renewal . But following an injury, a specific subset of facultative lung progenitors is triggered to initiate remodeling, a process that repairs the damaged tissue. When this mechanism is disrupted, healing fibrosis develops, which results in scarring, aberrant lung regeneration, and compromised organ function . For those with end-stage lung failure, lung transplantation (LTX) is still the only available treatment. Due to the limited number of tissue donors and viral contamination, an attempt has been made to construct the tissue using diverse cells and scaffolds, both natural and synthetic. This has resulted in the development of a new concept in science known as tissue engineering. As an academic field, tissue engineering has created a unique opportunity for the invention and refinement of therapeutic procedures for the treatment of congenital and acquired disorders, and its three basic pillars are scaffolds, growth factors, and cells. Scaffolds are a physical support and template for developing cells and tissues . Section title: 1 Introduction Educational score: 4.064188003540039 Domain: biomedical Document type: Review Language: en Bioengineered lung tissues are a new field of study that can help with this problem. While creating a fully functional bioengineered lung that might be transplanted is still difficult, new methods are being investigated to create ex vivo-engineered lung tissues that function and have the right gas exchange characteristics . Despite advancements in organ preservation methods, particularly ex vivo lung perfusion (EVLP), new approaches are desperately needed to close the therapeutic gap in biological (acellular) lung scaffolds and increase the number of transplantable tissues available . EVLP is a method that has revolutionized lung transplantation by improving the evaluation and use of donor lungs that might have been considered unfit, for transplant in the past. Before EVLP was introduced into practice numerous donor lungs with potential were rejected due to worries about their state leading to an organ utilization rate of 15–20 percent. Nevertheless, research indicates that approximately 40 percent of these declined lungs could be eligible for transplantation after undergoing further assessment, with EVLP. The importance of EVLP goes beyond evaluating lung viability as it plays a vital part, in revitalizing donor’s lungs that may be, on edge. Section title: 1 Introduction Educational score: 2.6523005962371826 Domain: biomedical Document type: Other Language: en Increased Utilization: EVLP has greatly expanded the pool of useable donor lungs by offering a platform for further evaluation, enabling transplant centers to use organs previously thought to be unsuitable. Section title: 1 Introduction Educational score: 3.2342116832733154 Domain: biomedical Document type: Other Language: en Decrease in Primary Graft Dysfunction (PGD): While PGD is still a risk, EVLP has been demonstrated to lessen its prevalence by enabling improved donor lung selection based on physiological data collected in real-time rather than only pre-transplant evaluations. Section title: 1 Introduction Educational score: 2.4541876316070557 Domain: biomedical Document type: Other Language: en Identifying Biomarkers: EVLP offers the chance to find biomarkers that are indicative of post-transplant results, which will allow donor selection procedures to be further improved. Section title: 1 Introduction Educational score: 2.7285959720611572 Domain: biomedical Document type: Other Language: en Therapeutic Interventions: According to recent research, pharmacological medicines or antibacterial therapies that are intended to enhance lung function before transplantation may be given during EVLP. Section title: 1 Introduction Educational score: 4.0330891609191895 Domain: biomedical Document type: Review Language: en Exosomes are nanovesicles that carry bioactive molecules and play an important role in cell-to-cell communication . They have important characteristics that make them superior to biological barriers and thus have a direct effect on various physiological and pathological processes . Based on research, it has been determined that exosomes can be an effective mechanism in tissue regeneration, especially in lung tissue regeneration . Also, in research, exosomes that are derived from certain cells or tissues have been investigated as a cell-free approach for the treatment of lung diseases, which promotes tissue repair and regeneration by promoting interactions between different lung cell lineages and facilitating paracrine-mediated bioburden transport . For this reason, investigating the mechanisms of using exosomes as potential therapeutic agents for lung regenerative medicine is particularly important in tissue engineering and regenerative medicine . In recent years, the use of exosomes in the clinic has increased significantly . Section title: 2.1 Bioactive exosome-loaded scaffolds Educational score: 4.175968647003174 Domain: biomedical Document type: Review Language: en Exosomes, also known as extracellular vesicles, play critical roles in a variety of biological processes, including cell proliferation, differentiation, and survival . They originate from a variety of sources, including stem cells and immune cells, and are enclosed in a bilayer membrane to safeguard their genetic material and proteins before being delivered to target cells . Exosome-containing proteins include membrane transport proteins, tetraspanins, biogenesis-related proteins, and heat shock proteins . Scaffolds enriched with exosomes promise to be a potential strategy in regenerative medicine, particularly for lung tissue engineering . These scaffolds mix exosomes, which are extracellular vesicles necessary for intercellular communication, with biomaterial scaffolds to aid in tissue repair and regeneration . The ability of exosome-loaded scaffolds to overcome the limitations of traditional cell therapies, like immune system rejection and decreased cell survival, is one of its key benefits . Section title: 2.1 Bioactive exosome-loaded scaffolds Educational score: 3.673846483230591 Domain: biomedical Document type: Other Language: en Exosome-loaded bioactive scaffolds, which can be made into three-dimensional structures like bioactive glasses, offer a special cellular environment that can boost exosomes’ therapeutic potential and encourage tissue repair . The use of bioactive scaffolds can improve tissue repair and regeneration by promoting cell adhesion, proliferation, and differentiation . Section title: 2.1 Bioactive exosome-loaded scaffolds Educational score: 4.114190101623535 Domain: biomedical Document type: Study Language: en To improve proangiogenic activity in bone healing, for instance, exosomes extracted from human bone marrow MSCs activated by dimethyloxaloylglycine were placed onto a porous hydroxyapatite scaffold . Similarly, exosomes extracted from human adipose-derived stem cells were combined with poly (lactic-co-glycolic acid) (PLGA)/polydopamine (pDA) structures to create a cell-free bone tissue engineering system . Exosomes were released from the scaffold slowly and continuously, which promoted MSC migration and greatly enhanced bone repair . Section title: 2.1 Bioactive exosome-loaded scaffolds Educational score: 4.131488800048828 Domain: biomedical Document type: Study Language: en Recent developments have focused on the use of specific biomaterials and scaffolds to stimulate cells to increase exosome production and on the integration of exosomes into 3D scaffolds with different architectures for implantation into damaged tissue . Researchers have also developed several methods to sustainably introduce exosomes into the environment after myocardial infarction . For example, exosomes produced from cardiomyocyte-derived induced pluripotent stem cells and encapsulated in hydrogel patches were directly delivered into infarcted rats’ hearts . Exosome patches increased ejection fraction, avoided cardiomyocytic hypertrophy, decreased ischemic injury, and improved heart repair . Bioactive scaffolds can enhance tissue repair and regeneration by stimulating cell adhesion, proliferation, and differentiation. Bioactive scaffolds can enhance tissue repair and regeneration by stimulating cell adhesion, proliferation, and differentiation . Wang et al. discovered that stem cell transplants with a biomimetic scaffold promote lung recovery. To alter lung tissue, they used a scaffold loaded with exosomes from mesenchymal stem cells (MSC) . Exosomes were able to recruit and differentiate MSCs, resulting in the production of functional lung tissue . Section title: 2.1 Bioactive exosome-loaded scaffolds Educational score: 3.93745493888855 Domain: biomedical Document type: Review Language: en Research by Yang and El Haj also highlighted the importance and value of designing and manufacturing scaffolds for cell and drug transfer, recognizing the need for biocompatible and biodegradable materials . Huang et al. also discussed the practical importance of exosome-filled scaffolds in the tissue regeneration and repair process and emphasized the important role of exosomes in cell communication and tissue regeneration . Additionally, Goldberg investigated most nanostructured materials for biotechnology and drug delivery and suggested using them to create exosome-loaded structures . Section title: 2.1.1 Role of exosomes in donor lung regeneration Educational score: 4.2328200340271 Domain: biomedical Document type: Review Language: en The therapeutic potential of exosomes in lung regeneration is supported by several studies demonstrating their ability to promote tissue repair, reduce inflammation, and enhance cellular function . Research have shown that exosomes derived from lung spheroid cells (LSC-Exo) exhibit superior therapeutic benefits compared to those derived from mesenchymal stem cells (MSC-Exo) in models of pulmonary fibrosis . Inhalation treatment with LSC-Exo significantly attenuated fibrosis induced by bleomycin and silica, restoring normal alveolar structure and decreasing the buildup of collagen and the growth of myofibroblasts appears to be a result of using exosomes in combating lung fibrosis. Moreover, exosomes have been shown to have inflammatory properties by influencing immune responses positively. In conclusion, Exosomes present an approach, to therapy with prospects, for improving the restoration of donor lungs. Their capability to enhance cell viability diminish inflammation and aid in tissue healing makes them crucial contributors to forthcoming approaches for lung ailments. Further exploration into the mechanisms and uses of exosomes will be crucial for creating treatments targeted at enhancing results for individuals, with impaired lung functionality. Section title: 2.1.1 Role of exosomes in donor lung regeneration Educational score: 4.085505485534668 Domain: biomedical Document type: Review Language: en Mesenchymal stem cell-derived exosomes can inhibit lung cancer cell proliferation and epithelial-mesenchymal transition . They could be achieved by delivering active cargoes such as miRNAs that directly target specific genes involved in tumor progression, such as MIER3 . Exosomes also regulate processes such as cell movement, cell proliferation, cell phenotype, and cell growth . They have properties such as anti-angiogenic and anti-inflammatory, which significantly affect the tissue regeneration process . In addition, they can transport active miRNA and release anticancer drugs, which inhibit the growth of tumor cells and cause cell death . Section title: 2.1.1 Role of exosomes in donor lung regeneration Educational score: 4.072418212890625 Domain: biomedical Document type: Study Language: en One of the challenges associated with exosome-based bioscaffolds for lung tissue regeneration is the need to improve large-scale production of exosomes to overcome limitations including short cycle time and low targeting capacity . In addition, another important challenge is the stability and storage conditions of exosomes, which should be taken into account for their correct conversion into the clinical setting . It is important to closely monitor the biodistribution and pharmacokinetics of exosomes due to factors such as their size and diversity . Finally, engineering alpha-6 integrin-expressing exosomes to target lung epithelial cells the incorporation of tissue-specific components into exosomes can improve their targeting ability and thereby affect transmigration . Section title: 2.1.1 Role of exosomes in donor lung regeneration Educational score: 3.9205615520477295 Domain: biomedical Document type: Review Language: en Exosomes have important clinical applications, including the potential to provide important information in the early detection of lung cancer . They also have important properties that make them promising therapeutic agents for diseases such as lung cancer because MSCs as well as their exosomes offer advantages for regenerative medicine such as immunosuppressive and non-immunogenic effects . Exosome-loaded scaffolds have also been thoroughly investigated in research for lung tissue regeneration and repair . Exosomes can also regulate the activity and proliferation of osteoclasts and make them suitable for the tissue regeneration process . Section title: 2.2 Cell-based scaffolds in lung regeneration Educational score: 3.894648313522339 Domain: biomedical Document type: Review Language: en Cell-based scaffolds represent a new approach to tissue engineering that integrates cells into biodegradable and biocompatible scaffolds to mimic the extracellular matrix . This mimicry provides structural support and biological, chemical, and mechanical signals that influence the development of new tissue . Although cell-based scaffolds have shown promise in several applications, including lung regeneration, they also present challenges, such as the need for a reliable source of human-sized scaffolds and a definitive sterilization method . Figure 3 . Section title: 2.2 Cell-based scaffolds in lung regeneration Educational score: 3.9782474040985107 Domain: biomedical Document type: Review Language: en Furthermore, stem cell transplantation based on biomimetic scaffolds has been shown to promote lung regeneration . A three-dimensional artificial MSC implant based on a biomimetic scaffold was constructed to create a favorable regenerative environment for lung tissue . This approach could help overcome the problems associated with the shortage of organ donors and avoid the need for lung transplantation . However, further research is needed to overcome the challenges associated with cell-based scaffolds, such as the need for a reliable source of human-sized scaffolds and a method for the final sterilization of the scaffolds . Advances in artificial intelligence and stem cell transplantation based on biomimetic scaffolds open promising prospects for the future of lung regeneration . Section title: 2.2 Cell-based scaffolds in lung regeneration Educational score: 3.9805257320404053 Domain: biomedical Document type: Review Language: en Recent advances in lung tissue engineering, including the development of smart biomaterials for scaffolds, and the use of on-chip organizational models to mimic the state of lung tissue, represent their important applications . The development of smart biomaterials for scaffolds has also had a significant impact on the field of lung regenerative medicine . These biomaterials actually activate certain tissue properties and can respond appropriately to changes in physiological conditions or external stimuli, in the same way that they have been used in various applications, including lung tissue repair and regeneration . For example, Balestrini et al. looked at removing cells from pig pleural membranes to create a tissue engineering alternative to lung tissue repair . In another study by Kim et al. , 3D artificial mesenchymal stem cell transplantation based on a biomimetic scaffold was used to create a positive regenerative niche for in situ lung regeneration from endogenous stem cells . Section title: 2.2 Cell-based scaffolds in lung regeneration Educational score: 4.118648529052734 Domain: biomedical Document type: Review Language: en Lab-on-a-chip models are useful tools for simulating lung tissue conditions, especially in lung reconstructive medicine . These models combine microfluidic systems and the cellular microenvironment to create constructs that mimic the structure and function of human organs and tissues while preserving dynamics . Lung-on-a-chip models are especially used to summarize lung function at the level of organs in laboratory conditions, where this feature allows lung-on-a-chip models to simulate the special microenvironment of the lungs . Lung diseases such as cancer, lung infections, asthma, and pulmonary edema can be studied using lung-on-a-chip models . In addition, bionic lungs, which are self-assembled three-dimensional functional units, provide another application for mimicking lung tissue conditions, helping to study diseases and evaluate drug efficacy . Cell removal techniques were also used to manufacture bioprostheses . The goal of this approach is to create patient-specific lungs for transplantation using cells derived from induced pluripotent stem cells . Section title: 3 Hydrogel-based exosome delivery system Educational score: 4.4850945472717285 Domain: biomedical Document type: Review Language: en ECM of the lungs has been mimicked by artificial polymeric . Scaffolds are crucial for tissue engineering (TE) development because they may be customized to imitate cellular microenvironments and provide structural and physicomechanical support, which can be used to moderate cellular physiology . Three-dimensional macromolecular polymer networks called hydrogels offer a three-dimensional microenvironmenthat improves exosomes’ therapeutic potential . They have some viscosity and elasticity and can be created chemically or physically through the use of cross-linking agents . The use of hydrogels in biomedical applications have attracted a lot of attention because of their structural and mechanical similarities to numerous tissues . A novel method of tissue engineering, especially for lung tissue regeneration, is represented by hydrogel-based exosome delivery devices . Exosomes are released by these systems in a regulated and controlled manner to maximize efficacy and reduce side effects. Exosomes are tiny extracellular vesicles that are loaded with certain lipids, proteins, and nucleic acids . They play a significant function in regulating intercellular communication and are exploited as transport vesicles for therapeutic applications . However, the stability and destiny of exosomes offer a substantial barrier to therapeutic uses, as they are degraded by the immune system immediately after injection in vivo . Therefore, the delivery method should be optimized to achieve high therapeutic efficacy and specificity, which means delivery of the desired exosomes to the target tissue . Biomaterials such as hydrogels allow exosomes to overcome poor tissue retention and provide a platform for controlled release to localize their activity . Section title: 3.1 Gelatin scaffold Educational score: 3.7889957427978516 Domain: biomedical Document type: Review Language: en Gelatin-based scaffolds have shown great promise in lung tissue engineering, as they mimic the natural ECM of lung tissue very well . These scaffolds, consisting of a gelatinous matrix, promote the growth and development of lung cells and therefore reflect the cellular microenvironment . This property makes gelatin-based scaffolds an ideal platform for research and the potential treatment of lung diseases . Section title: 3.1 Gelatin scaffold Educational score: 3.906921148300171 Domain: biomedical Document type: Study Language: en One way to improve the therapeutic potential of these scaffolds is to incorporate exosomes into the gelatinous matrix . Once implanted, these exosomes can be gradually released and provide cells with a continuous supply of nutrients and growth factors . This method is successfully used to regenerate bone tissue, demonstrating the potential utility of gelatin-based scaffolds in tissue engineering beyond lung tissue . Section title: 3.1 Gelatin scaffold Educational score: 3.9817636013031006 Domain: biomedical Document type: Study Language: en This gelatin scaffold exhibited desirable mechanical and degradation properties, flexibility, and cell adhesion . Although gelatin-based scaffolds are very promising, other challenges remain, such as ensuring the long-term stability of the scaffolds, maintaining the integrity of the gelatin matrix, and optimizing the release rate of exosomes . Further research and development are needed to overcome these obstacles and maximize the potential of gelatin-based scaffolds for the treatment of lung diseases. In general, gelatin-based scaffolds, alone or in combination with other materials, are very promising for lung tissue development . They have unique properties essential for optimal tissue function and have been shown to promote cell adhesion, proliferation and differentiation . Current research focuses on refining scaffold design and manufacturing and overcoming the challenges associated with integrating and controlling multiple nozzles in each cell . Further studies are needed to fully understand the behavior of cells seeded on the scaffold and how they might integrate into the recipient nervous system. More supplementary information is also provided in Table 1 . Section title: 4 Artificial lung scaffolds Educational score: 3.996324062347412 Domain: biomedical Document type: Study Language: en Artificial lung structures are three-dimensional structures designed to mimic the architecture and function of natural lung tissue . These structures are extremely important for tissue engineering and lung regeneration . They can be made from a variety of materials, including synthetic and natural polymers, and are designed to provide structural and physical support while mimicking the cellular microenvironment. One of the most essential aspects of these structures is their ability to renew the lung’s extracellular matrix (ECM) . The ECM is a complex network of proteins, lipids, and carbohydrates that provides structural support, controls cell development and differentiation, and facilitates cell-ECM interactions . Artificial lung scaffolds can create an appropriate environment for the growth and development of lung cells by utilizing the ECM function . Section title: 4 Artificial lung scaffolds Educational score: 4.212396621704102 Domain: biomedical Document type: Review Language: en Despite the potential benefits of artificial lung structures, their development also poses challenges . These include the identification of materials that mimic the ECM, provide physical support, and are biocompatible with the body . Other challenges include integrating cells into structures, maintaining the structural integrity of the structures, and preventing collapse . The development of artificial lung scaffolds is hampered by their heterogeneity and potential xenogeneic problems, making it difficult to scale up this technique in a reproducible and regulated manner . However, artificial or synthetic lung scaffolds, which typically use synthetic and natural polymers, offer an alternative . One of the advantages of using synthetic materials for scaffold fabrication is the ability to tailor their biological and physical properties to achieve a desired scaffold . Despite advances in lung tissue engineering, limitations remain . These include the complex branched structure of the pulmonary airways and the challenge of poor biocompatibility and haemocompatibility, i.e., the ability to be compatible with living tissue and blood respectively . Promising prototypes show that epithelialization and vascularization of grafts can be achieved using different methods . However, insufficient research has been conducted on 3D bioprinting and organoid techniques for parenchymal lung tissue . Figure 5 . Section title: 4.1 Hybrid scaffolds Educational score: 4.204544544219971 Domain: biomedical Document type: Review Language: en In lung tissue engineering, significant progress has been made in scaffold manufacturing, with a focus on hybrid bioactive scaffolds . These new scaffolds use a synthetic beta copolymer membrane with the ECM, creating a unique cellular microenvironment that supports tissue engineering in the lung . Hybrid scaffolds are critical for tissue engineering as they provide essential properties such as strength, elasticity, nutrient transfer, and cellular remodeling, all of which are vital for optimal tissue functionality . The use of hybrid materials in tissue engineering scaffolds in the lung is a growing area of research aimed at creating an optimal environment for tissue regeneration . The challenges and requirements for constructing such scaffolds are being resolved through interdisciplinary collaboration, and ongoing research focuses on perfecting scaffold design and manufacturing . An important innovation in this area is the use of hybrid materials to build an optimized lung structure . In this strategy, the favorable properties of two or more materials are combined to create a final scaffold that overcomes the limitations of each component . For example, the favorable properties of Acellular scaffolds for cell adhesion sites, organization, and differentiation signals combined with synthetic materials and advanced manufacturing techniques can lead to a desirable lung scaffold . Section title: 4.1 Hybrid scaffolds Educational score: 3.832620620727539 Domain: biomedical Document type: Review Language: en Despite the promising potential of these hybrid scaffolds, it must be recognized that they represent an ambitious goal that requires further scientific research and development . Ultimately, the goal is to create functional and biocompatible constructs for tissue regeneration, a challenge that must be addressed through in-depth research and experimentation . Furthermore, the integration of synthetic and natural materials into scaffolds not only promises to revolutionize the field of tissue engineering but also opens new avenues for the treatment of various lung diseases . Section title: 4.1 Hybrid scaffolds Educational score: 4.029465198516846 Domain: biomedical Document type: Study Language: en Lee et al. discovered that hybrid scaffolds that combine the mechanical strength of synthetic materials with the biological activity of natural materials have the potential to create an environment conducive to lung tissue growth and regeneration . Biodegradable polymers including poly (ε-caprolactone), polyethylene glycol, and poly (lactic-co-glycolic acid) are commonly used in hybrid scaffolds due to their mechanical stability and rapid decomposition. offers control . However, their natural fraction is frequently formed from extracellular matrix proteins and polysaccharides such as collagen, hyaluronic acid, and fibrin, all of which play critical roles in controlling cell activity and tissue integrity . Electrospinning and 3D printing were among the advanced fabrication techniques used to fabricate these hybrid scaffolds with a specific microarchitecture and precise porosity . Although progress is being made in this area, more research is needed to address challenges such as long-term biocompatibility. Section title: 4.1 Hybrid scaffolds Educational score: 3.7164595127105713 Domain: biomedical Document type: Review Language: en In general, over the past 2 decades, regenerative medicine has experienced significant technological advancements. Each milestone has introduced new methods that enhance tissue regeneration, scaffold design, and treatment efficiency. Table 2 outlines the chronological development of key technologies in this field, demonstrating how innovations have evolved to meet the growing demand for more effective and personalized therapeutic options. Section title: 5 Development of ex vivo transplantation Educational score: 3.4938838481903076 Domain: biomedical Document type: Other Language: en Transplantation of organs or tissues, outside the body, from a donor organism for evaluation and treatment before being transplanted into a recipient, is known as ex vivo transplantation. The process enables the assessment and improvement of organ viability and function before the actual transplant takes place to increase the likelihood of results. Section title: 5 Development of ex vivo transplantation Educational score: 4.22088098526001 Domain: biomedical Document type: Review Language: en In the field of lung regeneration, procedures like ex vivo transplantation are commonly used to evaluate donor lungs that may not meet transplant requirements by using methods such as EVLP ( Ex Vivo Lung Perfusion). This approach enables professionals to assess the viability and function of these compromised lungs by perfusing them with a solution under controlled conditions outside the body before transplantation is considered further. During EVLP procedures donor lungs are maintained at a temperature. Perfused with a nutrient-rich solution, like the Steen solution, to simulate blood flow. This method aids in improving lung function and evaluating factors, like oxygen levels and flexibility along with the feasibility . Ex vivo transplant methods notably expand the supply of donor lungs by reconsidering organs that were previously rejected for transplantation suitability purposes. Moreover, they allow for interventions during the perfusion stage, like medication administration or tissue repairs which enhance the graft quality before it is implanted into a recipient. The use of ex vivo transplant techniques has demonstrated outcomes in enhancing transplant success rates and lowering issues linked to graft dysfunction (PGG). As studies advance in this area, Ex vivo methods might develop more by integrating cutting-edge treatments to improve organ retrieval and enhance patient outcomes post-transplantation. Section title: 5 Development of ex vivo transplantation Educational score: 4.03939962387085 Domain: biomedical Document type: Study Language: en A basic problem confronting the area of tissue engineering is the inability to create perfusable microvasculature networks capable of supporting tissue viability and withstanding physiological forces without leakage . In small animal models, whole bioengineered lungs produced on acellular lung scaffolds were transplanted, but the lungs failed due to intravascular coagulation and endothelial barrier dysfunction, resulting in pulmonary edema . There has been no strategy that has allowed for the long-term survival of bioengineered lungs following transplantation . Section title: 5 Development of ex vivo transplantation Educational score: 3.9423201084136963 Domain: biomedical Document type: Study Language: en In vivo gas exchange is the main outcome of lung bioengineering research. Bioartificial lungs should be transplantable like cadaveric donor lungs, as the lung scaffolds maintain the vascular and airway architecture from the alveoli to the hilum. The viability of recellularized bioartificial lungs was first shown in a rat transplantation model . Section title: 5 Development of ex vivo transplantation Educational score: 4.113625526428223 Domain: biomedical Document type: Study Language: en To prepare for human clinical trials, it is necessary to scale up from small animals to clinically relevant large animals. To achieve this goal, Zhou H and colleagues repopulated pig lung scaffolds with human basal airway stem cells and umbilical vascular endothelial cells, resulting in human-sized bioartificial lung grafts. Using the pulmonary trunk and left atrial appendage as anastomoses, respectively, the pulmonary artery and vein were heterotopically transplanted into these grafts. One hour after surgical implantation, the graft continued to function and tolerate normal pulmonary blood flow . Section title: 5.1 Organoids in lung regeneration Educational score: 4.086021900177002 Domain: biomedical Document type: Review Language: en The term “organoid,” meaning “resembling an organ,” was first used by Smith et al., in 1946 to describe an instance of cystic teratoma . Organoids are three-dimensional, self-organizing are created by pluripotent or adult tissue stem cells. They provide an in vitro model for drug screening and replicate interactions between cells and niches in tissue formation, homeostasis, regeneration, and disease . A unique organoid culture system has been developed due to the technological advancements in in vitro culture technology over the past decade. Organoid cultures have now been adapted to cells of many different epithelial lineages . Section title: 5.1 Organoids in lung regeneration Educational score: 3.8279635906219482 Domain: biomedical Document type: Study Language: en Lung organoids have been successfully generated using adult stem cells, human pluripotent stem cells (hPSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). However, recent improvements in lung organoid systems have focused on organoid findings, particularly those from distal airway stem/progenitor cells . Section title: 5.1 Organoids in lung regeneration Educational score: 4.162137508392334 Domain: biomedical Document type: Study Language: en Many human lung airways are lined with pseudostratified epithelium, which is composed of various cell types including airway basal cells, secretory cells, ciliated cells, tuft cells, and neuroendocrine cells. Basal cells are present throughout the airways of the human lungs, including the small bronchioles with a diameter of 1 mm. These basal cells constitute approximately 30% of the pseudostratified lung epithelium and are firmly attached to the basal lamina. During homeostasis and repair, they can self-renew and produce secretory and ciliated luminal cells . Section title: 5.1 Organoids in lung regeneration Educational score: 4.261289596557617 Domain: biomedical Document type: Study Language: en Lung organoids can originate from several types of cells: basal cells, alveolar type II cells, and airway secretory cells. The genes that are expressed characteristically in basal cells are Integrin α6, p63, cytokeratin 5 (Krt5), and nerve growth factor receptor (NGFR) . The term “tracheosphere” refers to 3D organoids formed from trachea basal cells, and “bronchosphere” refers to organoids derived from bronchi or large airways According to Rock et al. the first tracheosphere was formed in a week in Matrigel using Krt5-GFPpos murine tracheal basal cells, and it had a visible lumen. After 14 days, p63pos Krt5/Krt14pos basal cells formed the outer layer of a pseudostratified epithelium, whereas Krt8pos ductal secretory cells and ciliated cells formed the inner layer . Section title: 6 Conclusions and future perspectives Educational score: 4.058977127075195 Domain: biomedical Document type: Review Language: en The extracellular matrix of the lungs has various specialized cell types with a distinct architecture that must maintain compliance during respiration, making them difficult organs to engineer. On the other hand, limited production and isolation of exosomes remains a major problem that must be addressed to fully realize the potential of these exosome scaffolds for regenerative medicine . Furthermore, the short lifetime of exosomes in tissue after in vivo implantation remains a major challenge for clinical applications . One approach to solving this issue is encapsulating exosomes in hydrogels which facilitate their ongoing diffusion into the damaged environment and thus improve their therapeutic effect . In conclusion, artificial lung constructs and hydrogel-based exosome delivery systems show promise for lung tissue engineering and regeneration . Types of bio-scaffolds for lung tissue regeneration are summarized in Table 1 . The above-mentioned scaffold in this review has numerous applications in lung bioengineering and will lead to advancements in future transplants. However, further research and development are needed to overcome the challenges associated with their use, particularly in terms of material selection and also exosome stability and release . Section title: 6 Conclusions and future perspectives Educational score: 4.007026672363281 Domain: biomedical Document type: Review Language: en Development of a suitable framework sufficient to fully clarify the physiological and biochemical interactions is necessary before proceeding with the creation of transplantable tissues. Procedures for decellularization and recellularization must also be devised. It is also necessary to study the short- and long-term effects of these transplantable tissues in animal models prior to clinical trials . Organoids represent a window of hope for lung regeneration. Lung organoids have proven to be an adaptable and useful tool for studying development, regeneration, homeostasis, and disease. Organoids are created by culturing different cells to represent chronic lung illnesses such as fibrosis and COPD. Future studies using newly separated cells from end-stage sick lungs will considerably increase our understanding of human lung illnesses, allowing for drug development and patient-specific treatment response monitoring. | Review | biomedical | en | 0.999996 |
PMC11695385 | Section title: Introduction Educational score: 0.969811737537384 Domain: other Document type: Other Language: en In the nineteenth century, London was the world’s financial centre, and a country’s ability to finance its trade and government was highly dependent on its access to the London money market (LMM) . However, as the global capital and trade markets became more interconnected and competitive in the late nineteenth century, some nations began questioning their financial reliance on London and looking for alternatives. One strategy was to establish a foreign banking presence, which had the capacity to provide informational and financial support to their businesses and commerce abroad and to provide alternatives to sterling as key trade currency. While research has demonstrated the benefits of foreign banks in supporting a nation’s trade and business overseas , attempts to break the dominance of sterling and the LMM were less successful . Section title: Introduction Educational score: 1.164969801902771 Domain: other Document type: Study Language: en This paper empirically examines the relationship between the LMM and the credit provision of non-British overseas banks in peripheral economies during the first wave of globalisation. Specifically, it studies whether fluctuations in the LMM influenced the credit supply of the German foreign bank Brasilianische Bank für Deutschland from its establishment in Brazil in 1889 until the outbreak of WWI. While the idea that the LMM affected the credit provision of non-British banks might not be novel, to the best of our knowledge, this is the first time this relationship is tested empirically and documented. We also show that this is causal using an instrumental variable (IV) approach that relies on the historical operation of the LMM. While the literature focuses chiefly on the market discount rate, we focus our attention on the spread between this rate and the floating rate, i.e. the rate at which London banks lent money overnight. We argue that the spread between the two is key for foreign banks that did not have a direct presence in London, and had to rely on bill brokers and discount houses through their London correspondents. Section title: Introduction Educational score: 1.0775303840637207 Domain: other Document type: Other Language: en In general, a larger spread signals that the conditions of the LMM are not stringent, i.e. the market is liquid and ample funds can be employed to rediscount bills. More in details, two principal mechanisms determined the link between the spread of the London market discount rate on prime bills (market rate) and the day-to-day loans rate (floating rate) and the credit supplied by foreign banks, which primarily used sterling-denominated bills of exchange to finance international transactions. The market rate reflects the price at which bills are bought and sold on the discount market, while the floating rate reflects the cost of short-term borrowed capital made available by London banks to banks and other agents, such as bill brokers and discount houses. The first mechanism involves London joint-stock banks. When the market rate is higher, it becomes more profitable for London banks to discount foreign bills. At the same time, low floating rates indicate that London banks have ample availability of funds to invest. As a result, demand for foreign bills in London increases when the spread between the market and floating rates is larger. The second mechanism involves bill brokers and discount houses, which play a key role in the discount market by intermediating between acceptors and final investors in bills of exchange by buying bills from the former and selling them to the latter 1 . These actors rely on narrow margins between the price of buying and selling bills for their profits, and therefore require large volumes of transactions to be profitable. Yet, their own capital is limited and most of their funds are borrowed from London bankers at the floating rate. A larger spread between the market and floating rate means that bills and borrowed money are relatively cheaper, allowing bill brokers and discount houses to intermediate larger amounts of bills. In some cases, discount houses do not re-sell the bills, but instead hold them until maturity. In this case, a larger spread between the market and floating rates means that they can borrow cheaply and lend at high-interest rates. Section title: Introduction Educational score: 0.9499775767326355 Domain: other Document type: Other Language: en The case of the Brasilianische is significant for several reasons. Firstly, it represents the importance of foreign banks in the internationalisation of Germany, a rapidly emerging economy at the time. By the turn of the century, it had become the second most important trade nation behind the UK and the third largest economy in the world 2 . German foreign banks were key to this successful expansion by providing financial services and informational assistance abroad . The Brasilianische is commonly acknowledged by coeval observers as a successful and representative blueprint of German overseas banking during the first globalisation . Secondly, the emerging economies of Latin America were a major destination for European foreign banking during this period, with foreign banks playing an essential role in the region’s economic development and integration into international trade markets 3 . According to Jones , Latin America was one of the markets where, after their first-mover advantage, British multinational banks faced harsher competition, particularly from German banks. Finally, the case of the Brasilianische highlights the competition faced by British banks from non-British banks, while also demonstrating London’s continued centrality in the global financial network. Despite attempts by German foreign banks to promote the independence of German international commerce from London and to offer the German mark as an alternative international currency, we find that they could not break away from the hegemony of the pound sterling. Section title: Introduction Educational score: 1.0532759428024292 Domain: other Document type: Other Language: en The Brasilianische Bank für Deutschland , founded in 1887 in Hamburg, aimed to facilitate trade relations between Germany and Brazil. It opened its first branch in Rio de Janeiro in 1889, providing direct credit and primarily using bills of exchange as a financing instrument. However, by statute, the bank was not allowed to use funds denominated in the Brazilian currency, the Milreis, for international business. To avoid exchange rate risks, it drew on European places that offered the most favourable conditions. As a result, over 80% of the bills of exchange it discounted were denominated in pounds sterling. Despite not having a branch in London, the bank had direct access to the LMM through its London agents and correspondent banks, and later through the London subsidiary of its mother institution, the Disconto-Gesellschaft . Section title: Introduction Educational score: 1.0283373594284058 Domain: other Document type: Study Language: en Using an OLS regression, we find a positive relationship between the monthly amount of credit lines authorised by the bank and the spread between the London market and floating rate. Our results suggest that the amount of credit authorised by the Brasilianische bank increases when there is (i) increasing demand for foreign bills in the London market relative, (ii) a decrease in borrowing costs for bill brokers and discount houses in London. Section title: Introduction Educational score: 1.558787226676941 Domain: other Document type: Study Language: en Our findings are not affected by reverse causality between our dependent and independent variables, as it is unlikely that the credit provision of the Brasilianische would impact London’s interest rates. However, our model may be subject to omitted variable bias. We include time-fixed effects and control for several additional variables to address this issue. We also employ an instrumental variable (IV) strategy to test the robustness of our results. Specifically, we use annual tax revenue collection in Great Britain and its effect on the spread of the market and floating rate as our IV. Individuals and companies based in Great Britain had to pay their annual income and other taxes at the end of March. Consequently, throughout the months of February and March, large amounts of money deposited at British joint-stock banks were withdrawn and transferred to the Government accounts at the Bank of England. This contraction in funds forced the joint-stock banks to reduce the amount of money they had available for daily loans. This led to an increase in the floating rate, and hence the spread decreased. As the Brasilianische Bank was not present in Britain and not impacted by British fiscal dynamics, we consider this shock to be exogenous. Our IV estimations support the findings of our OLS regression. Section title: Introduction Educational score: 0.9253780245780945 Domain: other Document type: Other Language: en The remainder of this paper is organised as follows. The next section gives the historical context describing the Brazilian economy in the late 19th and early twentieth century, the history of the Brasilianische Bank , and the operation of the LMM. Section 3 presents our empirical strategy focusing on our main model. Section 4 discusses our identification strategy and confirms our primary model’s results. The final section concludes the paper. Section title: The Brasilianische Bank in the Brazilian Economy Educational score: 1.0974735021591187 Domain: other Document type: Other Language: en Exports, primarily coffee and rubber, drove the Brazilian economy in the late 19th and early twentieth centuries 4 . Between 1889 and 1919, coffee comprised over 57% of Brazilian exports and 71% of the world’s total production. Rubber’s share in exports grew from 14.2 to 25.6%. The coffee industry faced a major setback due to overproduction and saturation in the international market, leading to a price drop. This drop decreased production between 1900 and 1905, until the Brazilian government stepped in as a direct buyer in 1905/6. Rubber exports also declined when Southeast Asian producers entered the market in the 1910s, accounting for only 5% of Brazilian exports in 1919 . Table 1 shows the export shares of the principal commodities of Brazil between 1870 and 1919. Table 2 shows the main trading partners of Brazil in 1910. Germany ranked third in terms of Brazilian exports and second in Brazilian imports. Table 1 Commodity export shares Brazil (% of total), 1870–1919. Source Abreu and Bevilaqua 1996 p. 9 Year Coffee Sugar Cotton Rubber Total 1870–79 56.3 11.8 9.7 5.5 83.3 1880–89 60.5 10.6 4.4 7.6 83.1 1890–99 65.4 6.1 2.5 14.2 88.2 1900–09 53.1 1.5 2.3 25.6 82.6 1910–19 52.1 2.4 1.7 16.4 72.6 Table 2 Brazil’s main trading partners . Source Dedinger and Girard Exports Imports USA 22,855,681 United Kingdom 13,676,221 United Kingdom 14,579,528 Germany 7,607,898 Germany 7,465,804 USA 6,127,582 France 5,309,449 France 4,539,270 Netherlands 3,241,507 Argentina 4,071,564 Trade flow in £ Pounds Section title: The Brasilianische Bank in the Brazilian Economy Educational score: 1.051710844039917 Domain: other Document type: Other Language: en These exports obviously needed to be financed, and the principal foreign actors in the Brazilian banking business were the British and the Germans . 5 The driving force of British banking engagement in the second half of the nineteenth century in Brazil was the increasing investment possibilities in the capital markets and infrastructure projects . The integration of the Brazilian economy into global markets made large-scale infrastructure work necessary. Mainly driven by coffee, the export boom triggered the construction of harbours and, most importantly, railway systems. The two largest and most influential German and British financial institutions in 19th-century Brazil were the Brasilianische Bank für Deutschland and the London and Brazilian Bank . The latter was the first foreign bank established in Brazil, opening its first branch in 1863 in Rio de Janeiro . By the beginning of the XX century, the British Bank of South America (firstly established in Brazil as the Brazilian and Portuguese Bank) had overtaken the London and Brazilian as the largest foreign bank in terms of deposits and credit (Table 3 ). The Brasilianische Bank für Deutschland was the second most important foreign bank. 6 Table 3 Deposits and lending of main banks operating in Rio de Janeiro . Source Retrospecto Commercial do Jornal do Commercio Bank Discounts and current accounts Deposits and creditor accounts Banco do Brasil 47,783,105 131,722,237 British Bank of South America 24,939,012 35,289,217 Brasilianische Bank fur Deutschland 23,735,277 22,454,431 Banco Commercial 12,087,988 13,583,182 Banco Mercantil do Rio de Janetro 8,162,655 3,406,308 London & River Plate Bank 6,708,650 12,706,099 Banco do Commercio 6,188,239 4,515,341 London & Brasillian Bank 5,414,998 15,985,422 Banco da Lavoura e do Commercio 3,735,859 1,990,006 Banco Espanol del Rio de la Plata 1,755,615 1,239,427 Figures in Milreis Section title: The Brasilianische Bank in the Brazilian Economy Educational score: 0.9956771731376648 Domain: other Document type: Other Language: en In 1887, the Disconto-Gesellschaft in Berlin and the Norddeutsche Bank in Hamburg founded the Brasilianische Bank für Deutschland . It opened its first branch in 1889 in Rio de Janeiro. Both banks, independently of each other, had already shown an interest in expanding to Latin American markets in previous years. While the Disconto was interested in entering Brazil’s infrastructure and railway construction business, the Norddeutsche had already been an important player in Brazil’s export and import sectors in previous decades . Yet, the risk and capital intensity had prevented an earlier market entry and ultimately led to the decision of Norddeutsche and Discontobank to combine their efforts in establishing a foreign bank. The focus of German banks on financing trade becomes even more evident when compared to their British counterparts. A direct comparison of the amounts of bills discounted by the Brasilianische Bank für Deutschland and the London & Brazilian Bank reveals that the German bank financed more bills . Section title: The Brasilianische Bank in the Brazilian Economy Educational score: 0.9984350800514221 Domain: other Document type: Other Language: en The Brasilianische Bank was the only German bank operating in Brazil until 1911, when the Deutsche Überseeische and the Deutsch-Südamerikanische Bank were established in Rio de Janeiro. In 1913, together these three banks possessed over nine branches in Brazil. The Brasilianische, however, was the only institution that exclusively concentrated its business on the Brazilian market. In the same year, three British banks with twenty-two branches were operating in Brazil; the London and Brazilian , the London and River Plate , and the British Bank of South America . Yet, the increasing competition from German banks had its impact. In 1906, British banks held some 77% of the foreign deposits in the major financial centres: In 1930, this figure was down to 31%. German banks ‘were by far the second most relevant actors in the region. (…) In terms of indicators such as total deposits, paid-in capital or profits, they were far bigger than their continental competitors, such as the French’ . Section title: The Brasilianische Bank in the Brazilian Economy Educational score: 1.0283457040786743 Domain: other Document type: Other Language: en The Brasilianische was closely linked to its mother institutions and the European money market. In 1887, the joint-stock capital of the Brasilianische was 2.5 Million mark, of which 1.5 Million were deposited at the Disconto-Gesellschaft and 1 Million at the Norddeutsche Bank . Furthermore, both mother institutions held most of Brasilianische’s stock shares. The bank’s headquarters, including the directorate and the supervisory board, were in Hamburg, Germany 7 , and every credit line granted by the Brasilianische had to be confirmed by the supervisory (Supervisory Reports Brasilianische). Section title: The Brasilianische Bank in the Brazilian Economy Educational score: 1.049066185951233 Domain: other Document type: Other Language: en The Brasilianische financed business in Europe and in Brazil in multiple currencies. 8 Yet, its use of domestic capital denoted in Milreis was restricted to finance business in Brazil only. This was the bank’s attempt to protect itself against the high volatility of the Brazilian currency and the resulting exchange rate risks . The capital to finance international business had to be acquired exclusively by drawing on Hamburg and Berlin, and, if more favourable conditions were available, on the international financial centres in foreign currencies . From the 1870s, Germany tried to establish the German Mark as an alternative trade currency in the international markets . However, throughout the entire observation period, on average, 80% per cent of the credit lines provided by the bank were denoted in the sterling pound ; hence, the bank’s focus was the LMM. The Brasilianische accessed European money markets via three main channels: (i) the bank held current accounts at its mother institutions that provided unlimited access to capital denoted in German Marks, (ii) the Disconto-Gesellschaft ’s London Office , and (iii) the bank’s London correspondents and agents gave access to the LMM (Table 4 ). Fig. 1 Monthly authorised credit lines of the Brasilianische Bank—bills discounting and direct credit—all currencies and in sterling pounds, 1889–1913 Table 4 Correspondents and agents of the Brasilianische in London Source The Banking Almanac and Directory and The Brazilian Review Bank Years active* Disconto-Gesellschaft 1901–1913 International of London Limited 1897–1904 N. M. Rothchild & Son 1898–1913 Union of London & Smith’s Bank Limited 1898–1913 William Brandt’s Sons & Co 1898–1913 Manchester and Liverpool District Banking Company Limited 1898–1913 We report 1897 because is the first year for which information is reported in the Banking Almanac. But even before 1897 the bank had correspondents in London Section title: The Brasilianische Bank in the Brazilian Economy Educational score: 1.0186796188354492 Domain: other Document type: Other Language: en The world’s financial centre at that time was the City of London. In 1912, the Brasilianische Bank reported: ‘London is not only the largest, but commonly also the cheapest discount market in the world. […] Even today, one has to admit—it would be disingenuous not to—the first thing you need to start an overseas banking business is (…) a drawing address in London’ 9 . Section title: The Brasilianische Bank in the Brazilian Economy Educational score: 1.0457379817962646 Domain: other Document type: Other Language: en The sterling bill of exchange, a financial instrument issued worldwide, was the main trade tool on the LMM . These became a liquid, secure short-term borrowing option, irrespective of their connection to actual transactions. The bill’s validity hinged on the acceptor and endorser(s)’ signatures, offering returns based on the market discount rate influenced by market conditions and the Bank of England’s policy. The Bank’s pledge to unrestrictedly convert sterling notes to gold underpinned the sterling bill’s safety, solidifying its central role in the LMM 10 . Section title: The Brasilianische Bank in the Brazilian Economy Educational score: 1.116692066192627 Domain: other Document type: Other Language: en Figure 2 provides a schematic representation of the functional roles of the actors in the LMM. Bills were drawn on London and sent there for acceptance. The role of acceptors was to screen the drawers of bills, i.e. to collect information on them and guarantee payment by accepting their bills. Once accepted, bills were bought by intermediaries, who had insider knowledge of the market and acted as screeners for acceptors and bills. These intermediaries usually did not hold the bills until maturity but endorsed them and re-sold them to deposit-taking institutions and the public. Deposit-taking institutions employed the funds collected among the public to make advances and buy bills from intermediaries. As a norm, they never rediscounted their bills but held them until maturity. Furthermore, deposit-taking institutions played another critical role in the LMM besides investing in bills. In fact, they also lent to the intermediaries most of the working capital they needed. Intermediaries borrowed short-term funds at the floating rate. When deposit-taking institutions had plenty of funds to lend, the floating rate was low, and vice versa. Fig. 2 Functional roles in the London money market Section title: The Brasilianische Bank in the Brazilian Economy Educational score: 1.0451170206069946 Domain: other Document type: Other Language: en Bill brokers and discount houses were unique intermediaries in the LMM acceptance market. Instead of costly direct engagement with merchant and individual acceptors, joint-stock banks outsourced bill scrutiny to these intermediaries . They evaluated the bill’s price based on the borrower’s solvency and liquidity risk, understanding that market conditions affecting acceptor and drawer could change the price without impacting solvency. Bill brokers and discounting house agents would visit the offices of banks interested in buying bills (usually London joint-stock banks) and the offices of banks interested in selling bills (usually foreign and colonial banks) multiple times a day. This way, they obtained fresh, first-hand information that would otherwise be costly for joint-stock banks. Section title: The Brasilianische Bank in the Brazilian Economy Educational score: 1.0254617929458618 Domain: other Document type: Other Language: en The market discount rate determined the price at which bills were bought and sold by bill brokers. The volume of bills the bill brokers transacted was a multiple of their capital, and they operated at leverage by borrowing short-term. 11 While discount houses also collected deposits from the public, bill brokers relied exclusively on borrowing at the floating market rate from London banks and Anglo-foreign and foreign banks. As fragile as this system might seem, it proved remarkably stable. As King 1972 , p.183) observed: ‘Then, as now, the call loan system rested upon the fundamental assumption that if one London banker were calling in loans, another banker would shortly receive a roughly equivalent amount, which he would seek to re-lend. In normal times, that was, and is, a warrantable assumption, and is the basic principle of deposit banking, as well as of bill dealing’. Section title: The Brasilianische Bank in the Brazilian Economy Educational score: 1.080313801765442 Domain: other Document type: Other Language: en In case of need, they could also sell their paper at a discount at the Bank of England—provided it was not a ‘Foreign Domicile’ or ‘Foreign Agency’ bill 12 . The floating rate hence determined how much leverage bill brokers and discount houses could take, and thus how large the turnaround of bills would be. When the discount rate was relatively high compared to the floating rate, bill brokers had all the incentives to move large amounts of bills. Furthermore, if market conditions were particularly favourable, they could even hold some bills until maturity. De facto , they would do maturity transformation by borrowing at the floating rate, and lending at the market discount rate. Therefore, when the spread between the discount and the floating rate was large it was easier for international banks to place their bills on the LMM. Another even more straightforward mechanism through which the spread could affect the demand for foreign bills was taking the perspective of the deposit-taking institutions. If the floating rate was low, it meant that these banks had an abundance of funds to employ. If the discount rate was high, it implied that investing in bills was more remunerative. Therefore, a large spread indicates that discounting bills was particularly profitable, relative to the liquidity of deposit-taking institutions. Section title: The Brasilianische Bank in the Brazilian Economy Educational score: 0.9941986203193665 Domain: other Document type: Other Language: en Figure 3 illustrates the fluctuations between the floating rate and the market rate against the backdrop of the Bank of England’s rate from 1890 to 1913. These fluctuations reflect the liquidity conditions in the LMM. A higher floating rate compared to the market rate suggests periods of liquidity shortage, prompting lenders to charge more for short-term funds. Conversely, a lower floating rate indicates liquidity surpluses, making it cheaper to borrow. The Bank of England’s rate acts as a benchmark, influencing these rates and signalling monetary policy changes. Contemporary commentaries acknowledged that the difference between the two rates reflected general liquidity conditions in the market. On Friday 19th October, floating rate was 1.5%, the market rate 3.75, and the BoE rate 4%. The ‘Discount and Loan Market’ section of the Economist commented: ‘money has remained plentiful’ 13 . By contrast, on Friday 9th March, day-to-day loans were 3.5%, market rate 4%, and BoE rate 4%—the Economist noted that ‘Money has grown scarcer’ 14 . Similarly, in October 1903, the Monetary Review of the Bankers’ Insurance Managers’ and Agents’ Magazine, described the September situation with the floating rate at 3.5 and the market rate at 4% as stringent. By contrast, the following month, when the floating rate was 1.75 and the market rate 3.65, the subtitle of the Monetary Review section was ‘easier money’ 15 . As shown in Fig. 4 (Sect. 4 ), the spread between the two rates is usually higher in autumn and lower in March. Peake observed these trends and explained that they were due to a greater supply of bills in autumn, necessitated by the financing of crop shipments after summer harvests. This situation affected the market rate more significantly than the floating rate. Conversely, February and March are quieter months for trade. Therefore, the stringent effect of the floating rate is not transferred to the market rate, as the supply of bills is low during this period. Fig. 3 Floating, market, and BoE rates Fig. 4 Market rate, floating rate and spread in London (monthly averages) Section title: Empirical Strategy: Credit Provision and the London Money Market Educational score: 2.2623069286346436 Domain: other Document type: Study Language: en In this section, we empirically investigate the relationship between the London spread of market and floating rate (London spread) and the monthly amount of credit authorised by the Brasilianische Bank between 1889 and 1913. Our main hypothesis is that the London spread positively affects the bank’s monthly credit lines. Expressed in the form of equation, the model is as follows: 1 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$X_{{\text{m}}} = \alpha_{0} + \beta {\text{LonSpread}}_{{\text{m}}} + \Upsilon_{{\text{m}}} + \lambda_{{\text{y}}} + \lambda_{{\text{m}}} + \varepsilon_{{\text{t}}}$$\end{document} X m = α 0 + β LonSpread m + Υ m + λ y + λ m + ε t where X m is the total value in sterling pounds of credit lines authorised by the Brasilianische in month m , α 0 is a constant. For this study, the key coefficient of interest is β , which shows the impact of the London spread on the credit lines of the Brasilianische . ϒ m is a set of control variables accounting for the different influences on the credit provision of the Brasilianische and/or the LMM conditions. The estimated model also includes year ( λ y ) and month ( λ m ) fixed effects. We use ordinary least squares (OLS) to estimate Eq. 1 , and we employ Newey–West standard errors to account for potential autocorrelation and heteroskedasticity. Section title: Empirical Strategy: Credit Provision and the London Money Market Educational score: 1.1903434991836548 Domain: other Document type: Study Language: en We take the data on monthly credit provided by Brasilianische Bank from 1889 until 1913 from Kisling . The data come from the official reports of Brasilianische Bank’s supervisory board (Aufsichtsratberichte) 16 . The supervisory board of Brasilianische Bank held its meetings three or four times annually at the bank’s headquarters in Hamburg, Germany. In these gatherings, the supervisory board determined the monthly credit lines for all branches of the Brasilianische Bank 17 , valid until the subsequent meeting. The duration of each credit line was at least the time between two board meetings, which, although variable yearly, was never less than one month. In the reports, financing was differentiated into: (i) lines of direct credit and (ii) the maximum value for the discount of bills of exchange. For this study, we are interested exclusively in the credit lines and bills of exchange denoted in sterling pounds. The monthly floating rate in London was taken from Nishimura , who retrieves the data from the Economist. London’s Monthly Market Discount Rate is taken from the NBER Macrohistory Database. For our econometric analysis, we test for the possible influence of a series of additional variables on the monthly credit authorised by the Brasilianische. Unless otherwise stated, we retrieved the information for these independent variables from the NBER Macrohistory Database 18 . For reasons of clarity, we maintained the original names of the variables as indicated in the source, which we report in italics. Table 12 in the Appendix shows the descriptive statistics of all variables employed. We test for the stationarity of the dependent and independent variables using the augmented Dickey-Fuller test. We use 12 lags to account for monthly data. Table 13 in the Appendix presents the results. At the 10% level, we can always reject the hypothesis that the dependent variable, our variable of interest, and our main control variables are non-stationary. They are thus considered I(0). Section title: Empirical Strategy: Credit Provision and the London Money Market Educational score: 1.333019733428955 Domain: other Document type: Study Language: en We include four main controls in our baseline regression. The variable ‘spread between discount and call money rates in New York’ quantifies the difference between the Commercial Paper Rate for New York and the Call Money Rate for USA . Its relevance stems from the US’s major trade partnership with Brazil and New York’s position as a significant financial centre, potentially influencing the dynamics of the LMM. The variable ‘Mark/$ exchange rate’ represents the Average Monthly Berlin Rate of Exchange on New York for Germany . It controls for fluctuations in the foreign exchange rate that might affect the investment decisions of the Brasilianische Bank. Furthermore, given the substantial involvement of German banks in the LMM, fluctuations in the value of the Mark can exert a notable influence on the dynamics of the LMM. The ‘spread between Reichsbank and Bank of England (BoE) official rates’ is the difference between the Official Bank Discount Rate for Germany and the Bank of England Policy Rate in the United Kingdom . Interest rate differentials between Britain and Germany could drive gold movements closely linked to LMM fluctuations. Additionally, this spread might influence credit limits in £, with the bank favouring credit lines in Marks if advantageous 19 . Finally, we consider the variable ‘Trade Union Members Unemployed in UK, % (log)’, which is the Trade Union Members Unemployed, Total for United Kingdom . It serves as an indicator of the state of the British economy, which at 8.22% of the global GDP in 1913 could have a significant impact on the LMM and the Brazilian economy 20 . Section title: Empirical Strategy: Credit Provision and the London Money Market Educational score: 2.9448113441467285 Domain: other Document type: Study Language: en Table 5 presents the results of our baseline regression ( 1 ). They confirm our main hypothesis. The coefficient of the London spread is positive and significant, confirming a positive correlation between the difference between the market and floating rate in London and the monthly amount of credit authorised by the bank. The effect is sizeable. A one-unit increase in the spread is associated with an average rise in monthly-authorised credit by 64,273 pounds, which is ~ 5% and ~ 20% of the mean and the standard deviation of our dependent variable, respectively (column 4). Table 5 Results regression estimations—baseline model—Eq. ( 1 ) (1) OLS (2) OLS (3) OLS (4) OLS Spread between discount and floating rates 75.767*** 65.801*** 71.054*** 64.273*** (23.170) (24.535) (18.782) (20.897) Spread between discount and call money rates, New York 19.959** 18.435* (9.722) (10.194) Mark/$ exchange rate − 1,685.396*** − 1,468.660** (499.661) (601.113) Spread between Reichsbank and BoE official rates − 26.927** − 23.289* (13.396) (12.642) Trade Union Members Unemployed in UK, % (log) − 93.210** − 53.216 (39.440) (44.533) Constant 290.345*** 218.145*** 7,404.490*** 6,406.049** (12.471) (27.574) (2,090.060) (2,539.114) Observations 300 300 300 300 Month indicators No Yes No Yes Year indicators Yes Yes Yes Yes Controls No No Yes Yes adjR2 0.825 0.827 0.830 0.829 F test model 873.2 1427 492.5 455.1 P -value of F model 0 0 0 0 The dependent variable is the total monthly credit authorised by the Brasilianische between 1889 and 1913 in thousand £. Standard errors are estimated with Newey–West and reported in parentheses *** p < 0.01, ** p < 0.05, * p < 0.1 Section title: Empirical Strategy: Credit Provision and the London Money Market Educational score: 1.649141550064087 Domain: other Document type: Study Language: en Column (1) shows the coefficient of London spread with only yearly indicators; Column (2) includes monthly and yearly indicators. Columns (3) and (4) add our main controls using only year and year and month indicators, respectively. Including monthly indicators ensures that seasonal patterns do not drive our results. The comparison of the results of our estimations with and without month indicators shows that seasonality is not likely to be the main driver of the correlation we find. In fact, the coefficient of the London spread is only marginally smaller when including monthly indicators. This is especially true in our models (columns 3 and 4), which include our main control variables. Section title: Empirical Strategy: Credit Provision and the London Money Market Educational score: 3.5851969718933105 Domain: biomedical Document type: Study Language: en To test the robustness of our findings, we run our estimations with two additional sets of control variables. Among these, only some variables are stationary at I(0), while others are at the I(1) level. In the latter case, we take the first differences (see Table 13 in the Appendix). If not otherwise stated, the information for these variables are taken NBER Macrohistory Database. The results are presented in Tables 6 and 7 , respectively. They confirm our main hypothesis, with the size and significance of the coefficient of London spread being consistent. Table 6 Results regression estimations—OLS—Eq. ( 1 )—including set of additional control variables (I) (II) (III) (IV) (V) (VI) Spread between floating and discount rates 67.626*** 62.749*** 70.622*** 63.972*** 71.731*** 64.524*** (18.813) (21.402) (18.594) (21.009) (23.113) (20.453) Spread between Reichsbank and BoE official rates − 26.893** − 24.130* − 25.207* − 21.690* − 26.591** − 24.528* (13.416) (12.845) (13.001) (12.696) (13.184) (12.748) Trade Union Members Unemployed in UK, % (log) − 86.797** − 47.746 − 87.403** − 49.015 − 89.067* − 50.565 (40.813) (46.852) (40.359) (44.840) (45.852) (44.071) Mark/$ exchange rate − 1,826.000*** − 1,548.534** − 1,805.641*** − 1,570.491** − 1,829.448*** − 1,570.000** (504.998) (614.893) (510.502) (612.729) (491.270) (620.751) Spread between discount and call money rates, New York 19.570** 19.523* 21.479** 20.593* 20.936** 18.941* (9.279) (10.688) (10.190) (11.141) (10.446) (10.566) Total Imports for Great Britain, mln £ = D , 2.546 3.790 (2.206) (3.523) Total Exports of Produce and Manufactures for Great Britain, mln £ = D , 2.967 6.687* (2.313) (3.413) Total exported bags of coffee from Rio de Janeiro (log) − 9.303 0.790 (27.339) (40.268) Price of coffee (log) £ = D , 260.514 310.023 (237.702) (237.309) Constant 8,000.697*** 6,749.458*** 7,913.535*** 6,832.133*** 8,128.750*** 6,822.006** (2,107.315) (2,595.265) (2,130.793) (2,587.250) (2,066.123) (2,697.258) Observations 299 299 299 299 299 299 Month indicators No Yes No Yes No Yes Year indicators Yes Yes Yes Yes Yes Yes Controls Yes Yes Yes Yes adjR2 0.825 0.824 0.825 0.824 0.825 0.824 F test model 639.7 421.4 606.4 454.3 470.5 333 P -value of F model 0 0 0 0 0 0 (VII) (VIII) (IX) (X) (XI) (XII) Spread between floating and discount rates 68.890*** 61.136** 58.242*** 55.844* 58.717*** 54.198** (18.671) (24.637) (17.318) (28.671) (16.768) (25.103) Spread between Reichsbank and BoE official rates − 25.532 − 22.832 − 23.238 − 22.877 − 23.685 − 23.369 (21.841) (20.548) (22.396) (21.477) (16.357) (15.049) Trade Union Members Unemployed in UK, % (log) − 87.193* − 50.303 − 109.562** − 69.587 − 107.193*** − 68.374 (47.865) (47.143) (45.902) (46.157) (39.483) (48.532) Mark/$ exchange rate − 1794.891** − 1500.934* − 1719.048** − 1544.421* − 1622.508*** − 1507.759** (706.150) (853.931) (733.403) (878.192) (563.611) (701.864) Spread between discount and call money rates, New York 21.080** 18.859 19.667* 17.635 19.517* 17.798 (10.385) (11.722) (10.556) (12.331) (10.789) (11.798) Pig Iron Output for Germany, ‘000 metric tons = D , − 0.137 − 0.222 (0.143) (0.233) Clearings of Reichsbank for Germany, bln Marks = D , 10.654 21.507 (19.340) (29.271) Earnings of Prussian − Hessian Railways from Freight for Germany, mln Marks = D , 0.801 1.458 (0.968) (1.221) Weights of Exports for Germany, ‘000 metric tons = D , 0.006 − 0.014 (0.016) (0.018) Weights of Imports for Germany, '000 metric tons = D , 0.012 0.005 (0.008) (0.005) Constant 7869.410*** 6546.134* 7984.943*** 7125.639* 7576.415*** 6982.474** Observations 299 299 273 273 273 273 Month indicators No Yes No Yes No Yes Year indicators Yes Yes Yes Yes Yes Yes Controls Yes Yes Yes Yes Yes Yes adjR2 0.824 0.822 0.664 0.661 0.665 0.660 F test model 158.4 146.7 111.1 77.48 315 221.4 P -value of F model 0 0 0 0 0 0 *** p < 0.01, ** p < 0.05, * p < 0.1 The dependent variable is the total monthly credit authorised by the Brasilianische between 1889 and 1913 in thousand £. Standard errors are estimated with Newey–West and reported in parentheses. The regressions are estimated with ordinary least squares (OLS) Table 7 Results regression estimations—OLS—Eq. ( 1 )—including set of additional control variables (I) (II) (III) (IV) (V) (VI) Spread between floating and discount rates 71.091*** 64.401*** 71.294*** 63.866*** 71.648*** 64.503*** (19.263) (20.525) (18.975) (20.943) (19.307) (21.488) Spread between Reichsbank and BoE official rates − 26.217** − 22.789* − 25.568* − 22.678* − 26.248** − 23.151* (13.084) (12.645) (13.499) (13.102) (13.022) (12.741) Trade Union Members Unemployed in UK, % (log) − 92.623** − 54.150 − 86.935** − 50.092 − 89.984** − 52.845 (40.588) (44.623) (40.853) (45.078) (41.171) (46.005) Mark/$ exchange rate − 1,819.396*** − 1,468.153** − 1,734.161*** − 1,466.511** − 1,806.717*** − 1,520.757** (522.510) (634.905) (501.548) (597.937) (518.249) (633.025) Spread between discount and call money rates, New York 22.035** 20.609* 21.007** 18.877* 21.031** 18.991* (9.784) (10.889) (10.049) (10.583) (9.945) (10.594) Security Price Index for London = D , 7.362 14.249* (7.328) (8.231) Milreis/$ exchange rate = D , − 19.725 − 18.668 (33.313) (33.352) Rubber prices = D , 35.814 35.649 (90.801) (79.654) Constant 7972.163*** 6397.796** 7613.656*** 6401.016** 7918.987*** 6629.293** Observations 299 299 299 299 299 299 Month indicators No Yes no Yes No Yes Year indicators Yes Yes Yes Yes Yes Yes Controls Yes Yes Yes Yes Yes Yes adjR2 0.825 0.824 0.825 0.823 0.824 0.823 F test model 585.4 465.1 620.8 503.7 581.9 465.9 P -value of F model 0 0 0 0 0 0 (VII) (VIII) (IX) (X) (XI) (XII) (XIII) (XIV) Spread between floating and discount rates 70.967*** 69.802*** 78.049*** 68.988*** 72.774*** 57.479*** 72.372*** 60.604*** (19.397) (21.583) (19.641) (21.532) (21.554) (22.115) (21.980) (22.965) Spread between Reichsbank and BoE official rates − 26.995** − 15.333 (13.346) (11.800) Trade Union Members Unemployed in UK, % (log) − 92.775** − 79.215* − 81.783** − 46.145 − 77.211* − 9.214 − 73.548* − 16.186 (42.161) (46.358) (40.803) (45.143) (44.685) (40.581) (42.631) (40.262) Mark/$ exchange rate − 1679.522*** − 1746.033*** − 2075.143*** − 1731.185*** (540.846) (665.038) (536.498) (583.098) Spread between discount and call money rates, New York 19.831* 22.300* 20.633** 19.147* 8.629 6.423 7.901 8.250 (11.912) (11.573) (9.839) (10.617) (10.485) (11.462) (11.938) (12.710) Spread between German official and market discount rate − 1.034 81.052 (28.949) (57.326) Spread between market discount rate in London and Berlin = D , − 3.342 8.632 (9.231) (11.163) Official discount rate Reichsbank 9.628 40.552** (17.792) (16.984) Market discount rate in Berlin 8.755 16.920 (17.190) (19.075) Constant 7380.768*** 7450.166*** 9040.184*** 7502.832*** 305.365*** 58.716 315.820*** 187.751*** (54.506) (69.514) (44.144) (46.357) Observations 300 300 299 299 300 300 300 300 Month indicators No Yes No Yes No Yes No Yes Year indicators Yes Yes Yes Yes Yes Yes Yes Yes Controls Yes Yes Yes Yes Yes Yes Yes Yes adjR2 0.830 0.832 0.824 0.822 0.826 0.830 0.826 0.827 F test model 486.8 268.8 750.5 537.3 1321 2021 1372 1968 P -value of F model 0 0 0 0 0 0 0 0 *** p < 0.01, ** p < 0.05, * p < 0.1 The dependent variable is the total monthly credit authorised by the Brasilianische between 1889 and 1913 in thousand £. Standard errors are estimated with Newey–West and reported in parentheses. The regressions are estimated with ordinary least squares (OLS) Section title: Empirical Strategy: Credit Provision and the London Money Market Educational score: 1.0894311666488647 Domain: other Document type: Other Language: en Since the primary function of the LMM was to finance international trade and the Brasilianische Bank did not only finance German trade, but also British firms, controlling for British imports and exports is crucial. We hence include in our first control set: ‘Total Imports for Great Britain, mln £ (difference)’, which is the first difference of Total Imports, Value for Great Britain ; ‘Total Exports of Produce and Manufactures for Great Britain, mln £ (difference)’, which is the first difference of Total Exports of Produce and Manufactures for Great Britain . To control for variations in Brazilian coffee exports, we include the ‘Total exported bags of coffee from Rio de Janeiro (log) (difference)’, the log value of the number of bags of 60 kg exported every month from Rio de Janeiro 21 . As the principal export commodity of Brazil, coffee had the potential to influence the lending of the Brasilianische . We furthermore account for possible demand shocks to the Brazilian economy caused by changes in the price of coffee with the variable ‘Price of coffee (log) £’, that is, Brazil Santos Arabicas Spot Price (Cents/Pound) (with GFD Extension) 22 . Section title: Empirical Strategy: Credit Provision and the London Money Market Educational score: 1.1295208930969238 Domain: other Document type: Study Language: en These controls are particularly important because we cannot include monthly indicators in our IV specification (see next section). Thus, including these variables should guarantee that we at least control for the seasonal fluctuations that should worry us the most—those in the coffee market. Finally, we test for the possible influence of the German business cycle. The Brasilianische funded trade between Germany and Brazil, and its lending likely depended on the macroeconomic conditions at home. At the same time, Germany was the second largest European economy 23 , and therefore its macroeconomic fluctuations inevitably could impact the LMM. We account for these influences with the following variables: ‘Pig Iron Output for Germany, ‘000 metric tons (difference)’ is Pig Iron Output for Germany ; ‘Clearings of Reichsbank for Germany, bln Marks (difference)’ is Clearings of Reichsbank for Germany ; ‘Earnings of Prussian-Hessian Railways from Freight for Germany, mln Marks (difference)’ is Earnings of Prussian-Hessian Railways from Freight for Germany ; ‘Weights of Imports for Germany, ‘000 metric tons (difference)’ is Total Imports—Weight for Germany ; ‘Weights of Exports for Germany, ‘000 metric tons (difference)’ is Exports, Total, Weight for Germany . 24 Furthermore, in Table 13 , we also control for imports and exports of the USA and France two other important trade partner of Brazil 25 . Section title: Empirical Strategy: Credit Provision and the London Money Market Educational score: 1.6410632133483887 Domain: other Document type: Study Language: en In our second set of control variables, Table 7 , we test for the possible influence of market conditions in Germany, dynamics in the British capital market, and fluctuations in Brazilian exchange rates. The ‘Spread between German official and market discount rate’ is the difference between the ‘Market discount rate in Berlin’, which is the Private Discount Rate, Prime Banker’s Acceptance, Open Market for Berlin, Germany, and the ‘Official discount rate Reichsbank’, which is the Official Bank Discount Rate for Germany. This spread serves as a crucial indicator of the tightness of credit conditions in Germany. As highlighted by Bignon et al. , a negative spread can signal that a Central Bank is rationing credit. Conversely, when the market rate significantly dips below the official rate, it implies an abundance of liquidity in the market, suggesting that banks do not rely heavily on Central Bank financing. Given that the Brasilianische Bank , headquartered in Hamburg, operates as a German bank, variations in the German rates and liquidity conditions can significantly influence its lending strategies. Moreover, considering Germany’s status as the second largest European economy after Britain, it is reasonable to presume that exogenous variations in German rates and market conditions could likewise impact the LMM. The variable ‘Spread between market discount rate in London and Berlin’ is the difference between Open Market Rates of Discount for London, Great Britain and Private Discount Rate, Prime Banker’s Acceptance, Open Market for Berlin, Germany . The rationale for their inclusion in the model is the same as for the ‘Spread between Reichsbank and BoE official rates’. ‘Security Price Index for London (difference)’ is Security Price Index for London, Great Britain . We control this variable to account for dynamics in the British capital market. While the effect on the LMM is straightforward, since many stock brokers borrowed short-term on the Money Market, we are concerned that a surging capital market could provide alternative investment opportunities for the Brasilianische Bank. We include ‘Milreis/$ exchange rate (difference)’ which is Brazil Real per US Dollar (with GFD Extension) to account for exchange rate fluctuations. 26 Lastly, it is essential to consider the heavy dependence of the Brazilian economy on rubber exports. A sudden shift in rubber prices could induce a demand shock within the Brazilian economy, potentially leading to repercussions in the LMM. To account for this, we include the variable ‘Price of rubber (log) $’ is Rubber Spot Price (USD/Kilogram) (with GFD Extension). 27 Section title: Empirical Strategy: Credit Provision and the London Money Market Educational score: 1.2053271532058716 Domain: other Document type: Study Language: en Our robustness checks reveal that none of these variables significantly alters the size and significance of the London spread coefficient. Demand shocks to the Brazilian economy, reflected through variables such as the total exported bags of coffee from Rio de Janeiro and the price of coffee, do not seem to exert substantial influence on the key relationship under study. This implies that factors intrinsic to the Brazilian economy, such as fluctuations in the coffee trade or the price of rubber, do not significantly interfere with the primary dynamics we are studying. Section title: Empirical Strategy: Credit Provision and the London Money Market Educational score: 0.8642358779907227 Domain: other Document type: Study Language: en Likewise, variables capturing the British trade, German business cycle, and German trade do not affect our key findings. Although these controls are integral to understanding the lending activities of the Brasilianische Bank and the macroeconomic conditions of these economies, they do not seem to change the central relationship being investigated. Similarly, variables related to the German discount rates, the spread between German official and market discount rate, the spread between the market discount rate in London and Berlin, the British capital market and the Brazilian exchange rate also fail to substantiate a significant shift in the size and significance of the London spread coefficient. This demonstrates that although these factors are vital to their respective economies, their influence does not materially alter our core findings. In sum, these robustness checks confirm the stability of our results. Section title: Empirical Strategy: Credit Provision and the London Money Market Educational score: 1.2227249145507812 Domain: other Document type: Study Language: en Finally, we conducted an additional set of robustness checks on our main explanatory variable, the London spread. The board meetings did not occur every month and were obviously not random. It is likely that the values of the spread between meetings influenced the decisions taken at these meetings. We introduced three alternative specifications of our London spread. The first specification takes the average of the spread between two meetings. The second specification carries forward the value of the spread of the last meeting until the following one. The third specification takes the average of the spread between t , t -1, and t -2. Our results remain robust, with our coefficient continuing to be large and significant. These results are displayed in Table 14 . Section title: IV approach: credit provision and tax revenues Educational score: 1.2144830226898193 Domain: other Document type: Study Language: en Our OLS regression analysis should not suffer from reverse causality between our dependent and independent variables. It seems quite implausible that a German foreign bank in Brazil had the capacity and market power to influence the European money market. However, despite the comprehensive robustness checks affirming our results, the possibility of omitted variable bias cannot be entirely disregarded. To mitigate potential endogeneity concerns, we are employing an instrumental variables (IV) approach, informed by our historical understanding of the functioning of the LMM. Section title: IV approach: credit provision and tax revenues Educational score: 1.1387652158737183 Domain: other Document type: Study Language: en Our IV is based on the historical effect of British tax revenues on the LMM. 28 The tax revenue collection in Great Britain at that time took place between February and April. During this period, British firms and individuals had to withdraw their money from London joint-stock banks to pay income and other taxes. The decrease in bank deposits resulted in the reduction of floating money, causing a contraction in short-term liquidity due to banks having fewer funds to extend as loans. This led to an increase in the floating interest rate from the end of February until the beginning of April, and consequently a decrease in the spread. We do not capture monthly tax payments. However, the tax payments were made directly to the Bank of England, leading to an increase in their public deposits. We use the fluctuations of these public accounts as IV 29 . Figure 8 in the Appendix reflects this relationship, illustrating the inverse correlation between the spread and the proportion of public deposits at the Bank of England (BoE). Section title: IV approach: credit provision and tax revenues Educational score: 1.2708829641342163 Domain: other Document type: Study Language: en The exogeneity of our IV derives from several factors. Firstly, it is based on the premise that the impact of British tax collection is confined to the banking sector within Great Britain itself 30 . The resultant fund transfers to the BoE’s Government account could only indirectly affect the Brasilianische’s credit lines through the LMM spread. Even the opening of a London branch by one of the mother institutions of the Brasilianische after 1901 (Disconto-Gesellschaft) would likely have had a limited impact. Typically, London branches of foreign banks didn’t collect a significant portion of domestic deposits, but rather focused on acceptances. It is hence highly unlikely that they had a significant amount of British customers that were subject to paying taxes and thus would withdraw their deposits. Secondly, there was the potential for unpredictable variations in the timing of tax payments. The income tax, being the primary tax collected, could be paid in two instalments—in March and July. While the bulk of it was typically paid in March, uncertainty remained for the bank regarding whether the entire amount would be paid in March or partly in July. Thirdly, the total income tax collected was not necessarily linked to the performance of the British economy. As depicted in Fig. 9 Appendix, the total income tax showed little correlation with GDP growth during the period under review. In fact, income tax rates experienced several changes during this time . This potential unpredictability further strengthens our argument for the exogeneity of the instrumental variable (IV). Section title: IV approach: credit provision and tax revenues Educational score: 1.0885249376296997 Domain: other Document type: Other Language: en Figure 4 displays the monthly average for the floating rate, the London market discount rate, and the spread between the two. It showed an evident contraction of the spread around March when the floating rate increased, and the market rate continued its trend. Contemporaries widely acknowledged this phenomenon. 31 Section title: IV approach: credit provision and tax revenues Educational score: 0.9695239067077637 Domain: other Document type: Other Language: en In his Academic study of the London money market, Peake displayed precisely the graph we plotted in Fig. 4 and commented ‘the rise in the floating rate to March is due, of course, to the collection of the taxes at the end of the financial year’. In his seminar study, The London Money Market, Spalding identified four periods of fluctuations in the LMM: 32 ‘This brings us to the second period, one in which the market is largely under the shadow of the tax-gatherers’ demand. Owing to the ingathering of revenue, stringent conditions are usually expected and experienced towards the end of the Government’s financial year in March. In fact, it is in March that the balances of the Chancellor of the Exchequer with the Bank of England reach their high-water mark and, as the money is kept off the market for a time, those who require accommodation have to pay high rates for it’. Section title: IV approach: credit provision and tax revenues Educational score: 1.3686214685440063 Domain: other Document type: Study Language: en One challenge our IV faces is the seasonality of the tax collection. It always happens during the same months of the year. However, the inclusion of monthly indicators in our estimation would absorb much of the correlation between our spread variable and our instrument. At the same time, the results of our OLS estimation show (Table 1 ) that the correlation between our dependent variable and the spread holds well with and without including monthly indicators, suggesting that seasonality should be a minor concern. However, a priori, we cannot rule out that the squeeze in the spread and the collection of taxes are a spurious correlation that depends on unobserved seasonal factors. We could not find any trace of such unobservable in the coeval press, but, fortunately, the constitutional crisis that followed the People Budget of 1909 allows us to dispel any doubt. In 1909, Lloyd George proposed a fiscal Budget with substantial progressive measures, including a land tax and a ‘super tax’ (or surtax) to be levied on incomes over £5000. The House of Lords vetoed the proposal, and new general elections were called. The Finance Bill was finally approved only in April 1910, and income tax collection for that year took place in May and June rather than in March. Figure 5 shows that the London spread and Government deposits typically show a robust opposite dynamic in February–March. In 1910, when tax collection took place in May–June, the same pattern emerged but shifted by three months. The absence of any pattern in February–March 1910 confirms that the strong divergence we observe in other years can be safely attributed to tax collection and not to unobserved seasonal patterns. Therefore, estimating our IV without month indicators should not be a problem. Fig. 5 London spread and Government deposits at BoE—Average 1889–1913 and 1910 Section title: IV approach: credit provision and tax revenues Educational score: 4.105062961578369 Domain: biomedical Document type: Study Language: en We use a 2SLS approach for our IV estimation. In the form of an equation, our model is expressed as follows: 2 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{LonSpread}}_{{\text{m}}} = \alpha_{0} + \zeta TR_{{\text{m}}} + \Upsilon_{{\text{m}}} + \lambda_{{\text{y}}} + \varepsilon_{{\text{m}}}$$\end{document} LonSpread m = α 0 + ζ T R m + Υ m + λ y + ε m 3 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$X_{{\text{m}}} = \alpha_{0} + \beta \widehat{{{\text{LonSpread}}_{{\text{m}}} + }}\Upsilon_{{\text{m}}} + \lambda_{{\text{y}}} + \varepsilon_{{\text{m}}}$$\end{document} X m = α 0 + β LonSpread m + ^ Υ m + λ y + ε m with (Corr (TR m ɛ m ) = 0). Section title: IV approach: credit provision and tax revenues Educational score: 2.4277446269989014 Domain: other Document type: Study Language: en Here, LonSpread is the spread between the London market and floating rate in month m . \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\widehat{{\text{LonSpread}}}$$\end{document} LonSpread ^ is the predicted value of LonSpread. TR m serves as our instrumental variable (IV), representing the percentage difference between the mean monthly public deposits at the Bank of England (BoE) and the mean annual public deposits at the BoE, measured relative to the mean annual public deposits at the BoE. λ y are year indicators. The rest of the specifications of Eq. ( 3 ) are identical to regression ( 1 ). Table 8 compares the results of our OLS baseline model with the results of our IV estimation. They confirm our previous findings. The coefficient of our main variable of interest, the London spread, is positive and significant. Our IV coefficients are larger than our OLS coefficients, but this should not be due to a weak instrument problem. The first stage IV F statistics (reported in Table 8 ) confirms that our instrument is very strongly correlated with our dependent variable, and thus we can rule out the issue of a weak instrument. Table 8 Results regression estimations—IV model—Eqs. ( 1 ) and ( 3 ) (1) OLS (2) IV (3) OLS (4) IV Spread between floating and discount rates 75.767*** 95.472** 71.054*** 97.032** (23.170) (43.448) (18.782) (46.916) Spread between discount and call money rates, New York 19.959** 16.657* (9.722) (8.691) Mark/$ exchange rate − 1685.396*** − 1799.487*** (499.661) (510.990) Spread between Reichsbank and BoE official rates − 26.927** − 21.684* (13.396) (11.421) Trade Union Members Unemployed in UK, % (log) − 93.210** − 97.649** (39.440) (39.183) Constant 290.345*** 282.069*** 7404.490*** 7878.006*** (12.471) (19.499) Observations 300 300 300 300 Month indicators No No No No Year indicators Yes Yes Yes Yes Controls No No Yes Yes R -squared 0.839 0.846 adjR2 0.825 0.825 0.830 0.829 F test model 873.2 492.5 P -value of F model 0 0 0 0 First stage IV F -stat 103.1 69.89 The dependent variable is the total monthly credit authorised by the Brasilianische between 1889 and 1913 in thousand £. Standard errors are estimated with Newey–West and reported in parentheses. Column 1 shows the result of our OLS baseline model without additional control variables, while column 3 includes these variables. Columns 2 and 4 present the results of the respective IV regressions, without and with control variables *** p < 0.01, ** p < 0.05, * p < 0.1 Section title: Concluding remarks Educational score: 0.7646663188934326 Domain: other Document type: Study Language: en This article studies the role of the LMM in determining the credit supply of non-British international banks in peripheral economies during the first wave of globalisation. At that time, London was the global financial hub, and sterling was the key international trade currency. Research and coeval studies have illustrated the importance of foreign banking in financing foreign trade of British competitors, such as Germany. At the same time, literature affirms that attempts to break the dominance of sterling and the LMM have failed. Yet, there seems to be a lack of quantitative research on the impact of the London Market dominance on the financial capabilities of non-British banks abroad. Section title: Concluding remarks Educational score: 1.001563310623169 Domain: other Document type: Other Language: en Using the example of the German bank Brasilianische Bank für Deutschland in Brazil between 1889 and 1913, we find that the monthly credit lines authorised by the bank were positively related to the spread between the London market and the floating rate. Our findings suggest that the bank increases the provision of credit when there is either a rise in demand for foreign bills in the London market or a decrease in borrowing costs for bill brokers and discount houses in London. Even without a branch in London, the Brasilianische was able to benefit from the LMM thanks to its network of correspondents and agents, and, since 1901 from its mother institution, the Disconto-Gesellschaft . Section title: Concluding remarks Educational score: 1.6106784343719482 Domain: other Document type: Study Language: en We provide robust evidence that this relationship is causal. Firstly, we exclude possible issues of reverse causality since it is implausible that the Brasilianische had the capacity to influence conditions in the LMM. Secondly, the introduction of a large set of control variables that account for potential confounding effects does not change the results of our econometrical analysis. Thirdly, we develop an identification strategy (IV) based on the effect that the annual tax collection in Great Britain had on the liquidity conditions in the LMM and on the spread between the market and floating rate. Since the Brasilianische Bank was not subject to British taxes, we argue that this shock is exogenous. Section title: Concluding remarks Educational score: 0.9907456636428833 Domain: other Document type: Other Language: en These empirical results offer important new evidence and insights on the dynamics of the rising competition of economies challenging Great Britain’s global financial hegemony before WWI. It broadens our understanding of the development of financial centres, financial institutions, and their interdependence. | Other | other | en | 0.999996 |
PMC11695401 | Section title: Introduction Educational score: 4.333411693572998 Domain: biomedical Document type: Review Language: en Major depressive disorder (MDD) is a severe condition acknowledged worldwide as a major factor for disability and functional impairment ( 1 ). Adolescence and young adulthood are peak periods for the onset of major depressive disorder (MDD) ( 2 ). Early-onset depression negatively impacts physical and mental development and is associated with poorer academic, occupational, and social achievements ( 3 ). Furthermore, depressive episodes during adolescence are linked to an increased risk of suicide, psychiatric and medical comorbidities, as well as a heightened risk of major depressive and anxiety episodes later in life ( 4 ). Suicide has been identified as the second most prevalent cause of death among adolescents with MDD ( 5 ). To mitigate the impact of depression among young individuals, it is essential to select treatments that are both effective and safe, while also identifying vulnerability factors early in MDD to inform targeted prevention and early intervention strategies ( 6 , 7 ). Despite the proven effectiveness of pharmacotherapy and psychotherapy ( 8 ), challenges remain with the use of pharmacotherapy in clinical practice ( 9 ). These include poor acceptance by parents, low medication adherence, limited drug choices for adolescents, and the potential risk of suicide associated with the use of anti-depressants ( 10 ). Although the therapeutic benefits of modified electroconvulsive therapy (MECT) have been reported, there is still a lack of comprehensive research on its application in adolescent patients ( 11 ). The efficacy, risk-benefit ratio, and ethical considerations regarding informed consent for MECT in this population are the subject of ongoing controversy ( 12 ). Consequently, there is a pressing need to explore alternative treatment approaches that are both safe and effective in improving depressive symptoms in minors with MDD. Section title: Introduction Educational score: 4.549559593200684 Domain: biomedical Document type: Review Language: en Repetitive Transcranial Magnetic Stimulation (rTMS) is a non-invasive therapeutic approach. rTMS is based on the physical principle of electromagnetic induction, whereby a magnetic field is generated through a transcranial magnetic stimulation coil. This leads to the production of an induced electric field within the cerebral cortex that is sufficient to elicit action potentials in neurons within the targeted brain region. In 2008, the Food and Drug Administration (FDA) approved application of 10 Hz high-frequency rTMS to the left dorsolateral prefrontal cortex (DLPFC) in adults with treatment-resistant depression ( 13 ). The DLPFC is primarily associated with negative thought patterns and feelings of hopelessness. Dysfunction in this region is an integral part of the pathophysiology of MDD and may lead to deficits in negative cognitive regulation ( 14 ). The activity of the DLPFC is often suppressed in patients with depression. Numerous clinical studies have demonstrated that rTMS at this site can help to restore the function of DLPFC, thereby improving mood and depressive symptoms ( 15 ). In addition to improving depressive symptoms, some studies have also shown that rTMS on the DLPFC can significantly decrease suicidal ideation in MDD patients ( 16 ). Several systematic reviews have concluded that traditional rTMS based on the 5-cm positioning method is a valuable therapeutic option for adolescent depression ( 17 , 18 ). A series of high-frequency rTMS is effective for the treatment of depressive disorders in adolescents, while also significantly reducing SI. Zhang ( 19 ) reported this treatment has superior anti-depressant efficacy in adolescents compared to adults. In the evaluation of stimulation parameters, the study by Wall indicated that applying 10Hz rTMS at 120% of the motor threshold to the left DLPFC can effectively reduce the severity of depression in adolescents, without serious adverse effects ( 20 ). High-frequency rTMS can alter the inhibition-excitation imbalance associated with MDD in the cortical and limbic brain areas. This results in better efficacy against depression and reduced SI, with good tolerance and no significant adverse reactions ( 21 ). Section title: Introduction Educational score: 4.216452598571777 Domain: biomedical Document type: Study Language: en The clinical efficacy of rTMS is known to be influenced by stimulation parameters such as the target site and the intensity, duration, and frequency of stimulation ( 22 ). The most commonly used strategy to position the DLPFC target site is to place the TMS coil 5 cm anterior to the primary motor cortex representing the hand, with measurement along the scalp curvature. This is known as the 5-cm rule. However, this method may be inaccurate for several reasons ( 23 ). The use of an absolute distance such as 5 cm introduces significant bias related to head size, since larger heads have a greater distance between the motor cortex and the DLPFC. Herwig et al. showed that the “5-cm rule” accurately positions the DLPFC target site in only about 30% of cases, and often positions the site behind the DLPFC ( 24 ). Structural Magnetic Resonance Imaging (sMRI)-based rTMS effectively addresses the inaccuracy of the surface 5-cm positioning method and has been adopted for head positioning treatment in adult MDD patients. A systematic review found that sMRI-guided TMS treatment gave a response rate of 15% to 83% in MDD adults, while also reducing SI ( 25 – 27 ). However, studies on MRI-guided rTMS treatment are very limited for adolescents. Suicidal behavior in adolescent patients underscores the need to explore more optimized treatment plans in clinical settings ( 28 ). With this background in mind and based on high-frequency rTMS positioning in MDD adults, we evaluated the efficacy of sMRI-guided positioning combined with pharmacotherapy for the treatment of depressive symptoms and SI in MDD adolescents. This approach was compared to surface 5-cm-guided rTMS positioning combined with pharmacotherapy, and pharmacotherapy alone. The results of this study provide new insights for the individualized treatment of MDD adolescents. Section title: Inclusion criteria Educational score: 4.107609748840332 Domain: biomedical Document type: Study Language: en From April 2022 to December 2023, MDD patients were recruited at the Hangzhou Seventh People’s Hospital according to the following inclusion criteria: (1) age 13-18 years (inclusive); (2) meet the criteria for current MDD according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV), as assessed by two professional psychiatrists using the Mini International Neuropsychiatric Interview (MINI); (3) Han ethnicity; (4) right-handed; (5) score of ≥14 on the 17-item Hamilton Depression Rating Scale (HAMD-17); (6) score of non-zero for items four and five of the Beck Scale for Suicide Ideation-Chinese Version (BSI-CV); (7) no medication taken for two weeks prior to enrollment, and only one type of SSRIs anti-depressant used after enrollment, with no change in the type of medication, although dosage adjustments were made based on clinical conditions. The exclusion criteria were as follows: (1) contraindications for MRI, such as metal implants; (2) complication with severe somatic disease, including inflammatory diseases and autoimmune diseases; (3) current or past neurological diseases or history of brain trauma; (4) history of manic or hypomanic episodes. Upon enrollment, all patients must undergo the Structured Clinical Interview for DSM (SCID) assessment. This study was approved by the Ethics Committee of the Hangzhou Seventh People’s Hospital [ethics approval number (078) of 2022]. The study followed the guidelines of the Declaration of Helsinki. Prior to the study, all participants and their parents or legal guardians provided written informed consent. Section title: Enrollment process Educational score: 3.967355251312256 Domain: biomedical Document type: Study Language: en All enrolled MDD adolescents were allocated of their own free will into one of three groups with open labels: sMRI-guided positioning combined with pharmacotherapy (sMRI navigation group), surface 5-cm positioning combined with pharmacotherapy (5-cm positioning group), and pharmacotherapy alone(pharmacotherapy group). All participants in the sMRI navigation and 5-cm positioning groups received rTMS treatment once per day and five times per week (total of 10 rTMS sessions over 2 weeks) in conjunction with pharmacotherapy, while the pharmacotherapy group received only the pharmacotherapy regimen. The type of anti-depressant medication taken during the treatment period was not altered, with the medication dosage adjusted according to the clinical condition. In addition, patients with sleep disorders were also given benzodiazepine-class sleep aids. Section title: sMRI precise positioning Educational score: 4.1263227462768555 Domain: biomedical Document type: Study Language: en Patients received an MRI scan at baseline. MRI data were acquired using the MAGNETOM Prisma 3.0T MRI system and included three-dimensional (3D), high-resolution T1-weighted images and dynamic imaging of brain function of the scalp and 1-mm brain slices. Individual MRI structural and functional images were used to build the 3D model and to calculate the TMS target area. 3D spatial coordinates of individual target areas were derived using brain region templates. The relative position of TMS on the brain was determined by deep learning and image registration with the help of a navigational 3D camera and TMS locator. The TMS target site and TMS coil can be monitored visually after 3D rendering, and the coil focus can be oriented in real-time to the target site. This enables precise navigational positioning and TMS treatment . Section title: rTMS protocol Educational score: 4.13227653503418 Domain: biomedical Document type: Study Language: en This study utilized a RT-100 magnetic field stimulator (Sichuan Junjian Wanfeng Medical Equipment) equipped with an integrated liquid cooling system (internal circulation) and a figure-eight coil (model CB085A, coil specification 85 mm). The resting motor threshold (RMT) for each participant was determined according to standard clinical practice. The stimulation site was the DLPFC, with the sMRI navigation group targeting the MNI coordinates (-26; 44; 49), and the 5-cm positioning group targeting 5 cm anterior to the left M1. RTMS was conducted at 90% RMT intensity and 10Hz for a total of 2400 pulses. This included a sequence of 4 seconds on and 16 seconds off, for 19 minutes and 44 seconds. The treatment comprised 10 sessions, with one session per day. RMT was defined as the minimum rTMS intensity required to induce a motor response in the contralateral resting abductor pollicis brevis muscle. Section title: Clinical assessment Educational score: 4.138955593109131 Domain: biomedical Document type: Study Language: en The HAMD-17 scale and subscale score was used as the primary outcome measure and was assessed at baseline, after 5 sessions of rTMS (1 week), and after 10 sessions of rTMS (2 weeks). The BSI-CV and subscale was used as the secondary outcome measure and includes the intensity of SI and the risk of suicide. BSI-CV was also assessed at baseline and after 1 week and 2 weeks of rTMS. The results for the timeline “at the most despondent, most depressed” in the BSI-CV scale were almost identical to those prior to treatment and thus did not show a treatment effect. Data from the second assessment were therefore analyzed on the “past week” timeline. Taking into account the impact of medication before and after treatment, we used changes in medication dosage as a covariate. Based on the literature ( 29 ), we equated 100 mg of sertraline to 20 mg of fluoxetine and 10 mg of escitalopram. This study also compared the treatment efficacy among three groups of MDD adolescents, where treatment responders or those with effective treatment were defined as individuals with a reduction of 50% or more in HAMD-17 scores post-treatment. Section title: Statistical analysis Educational score: 3.967200756072998 Domain: biomedical Document type: Study Language: en Statistical analyses were conducted using SPSS 27.0 (IBM, USA) and GraphPad Prism 7.0 (GraphPad Software). Categorical variables were presented as frequency and proportion, with comparisons between groups were made using the chi-square test. Continuous variables with a normal distribution were reported as means and standard deviations. A two-way repeated measures ANOVA was used to analyze main and interaction effects across three treatment groups and three time points for all outcomes. Additionally, the difference scores from baseline were assessed using the same method. Post hoc pairwise comparisons were performed with Bonferroni adjustments for multiple comparisons, applying a significance level of α=0.05. The Bonferroni-adjusted significance level for each comparison was set at 0.016. Section title: Clinical and behavioral characteristics Educational score: 4.10677433013916 Domain: biomedical Document type: Study Language: en All patients met the preliminary diagnosis of MDD. A total of 130 patients were screened, of which four patients failed the screening, two failed to complete the MRI examination, and one withdrew from the study due to early discharge after screening. Of the final 123 enrolled patients, 44 were complicated with anxiety disorders, one with obsessive-compulsive disorder, and one with attention deficit disorder. The final analysis included 33 patients in the sMRI navigation group, 41 in the 5-cm positioning group, and 49 in the pharmacotherapy group. With all three groups, assessments were carried out at baseline and after the first and second weeks of enrollment. The three groups showed no significant differences (P > 0.05) in baseline clinical characteristics such as age, gender, education level, disease duration, and family history of mental illness ( Table 1 ). All enrolled patients took only one kind of anti-depressant medication during the rTMS period, with 113 taking Selective Serotonin Reuptake Inhibitors (SSRIs), 74 taking sertraline (dosage range 50-150 mg), 31 taking fluoxetine (dosage range 20-60 mg), 18 taking escitalopram (dosage range 10-20 mg), and 75 patients taking benzodiazepine sleep medication in addition. Section title: Main effect test of HAMD total score and its factor score Educational score: 4.144658088684082 Domain: biomedical Document type: Study Language: en Normal and variance homogeneity tests were performed on the total score and factor scores of HAMD in the 3 groups at different time points during treatment. The data that met the normal test were tested by two-way repeated measures ANOVA. After 2 weeks of treatment, the HAMD scores and their subscale scores were all decreased in the sMRI navigation group, the 5-cm positioning, and the pharmacotherapy group compared to baseline. All emotional scores and their subscale scores were significantly reduced as the corresponding treatments progressed . There was significant interaction between time and grouping in HAMD score (F=11.223, p<0.001), somatic/anxiety factor (F=4.151,p<0.001) and retarding factor score (F=7.262, p<0.001).These results indicate that the time factor has different effect on the emotional state of patients with different treatment methods. Therefore, the time effect and the intergroup effect were tested separately for the three groups of patients. Section title: Individual effect test of HAMD total score and subscale scores Educational score: 4.166860103607178 Domain: biomedical Document type: Study Language: en Two-way repeated measures ANOVA was used to compare the changes in HAMD scores and subscale scores at baseline, 1 week and 2 weeks within the three groups. There were no statistically significant differences in the total HAMD scores and its subscale scores among the three groups of patients at baseline (P>0.05). The total HAMD score and its subscale scores were compared within each of the three groups, showing significant differences: baseline > 1 week after treatment >2 weeks after treatment (P<0.001). At the end of the first week of treatment, the total HAMD scores in the sMRI navigation group were lower than those in the 5-cm positioning group (P <0.001) and pharmacotherapy alone group (P <0.001), and the scores in the 5-cm positioning group were lower than those in the pharmacotherapy alone group (P <0.001). At the end of the first week, the sMRI navigation group had lower anxiety/somatization scores than both the 5-cm positioning group and the pharmacotherapy alone group (P<0.001 for both), and the 5-cm positioning group scored lower than the pharmacotherapy alone group (P <0.001). For the retardation score, both the sMRI navigation group and the 5-cm positioning group scored lower than the pharmacotherapy alone group (P <0.001), but there was no significant difference between the sMRI navigation group and the 5-cm positioning group (P =0.092). At the end of the second week, the sMRI navigation group had lower total HAMD and anxiety/somatization scores than both the 5-cm positioning and pharmacotherapy alone groups (P <0.001 for all comparisons). The 5-cm positioning group also scored lower than the pharmacotherapy alone group (P <0.001). For retardation scores, both the sMRI navigation and 5-cm positioning groups scored lower than the pharmacotherapy alone group (P <0.001), but there was no significant difference between the sMRI navigation and 5-cm positioning groups (P =0.086). More details can be found in Table 2 and Figure 2 . Section title: Comparison of antidepressant treatment efficacy in MDD patients Educational score: 4.160578727722168 Domain: biomedical Document type: Study Language: en The proportion of treatment responders in the sMRI navigation group reached (57.57%, 19/33) in the first week, which was higher than that in the 5-cm positioning group (31.70%, 13/41) and the pharmacotherapy group (20.0%, 10/49), showing a significant difference in treatment efficacy among the three groups (p=0.002). In the second week of treatment, the proportion of treatment responders in the sMRI navigation group increased to 84.84% (28/33), which was higher than that in the 5-cm positioning group (60.97%, 25/41) and the pharmacotherapy group (46%, 23/49), with significant differences in treatment efficacy among the groups (p=0.002). Further comparison revealed significant differences in the reduction rates of the retardation subscale scores (p<0.001) and the somatization/anxiety subscale scores (p<0.001) among the groups in the first week. In the second week, significant differences were found among the groups [retardation subscale (p<0.001); somatization/anxiety subscale: (p<0.001)]. Post-hoc pairwise tests revealed that the differences in reduction rates primarily stemmed from comparisons between the sMRI navigation group with the 5-cm positioning and pharmacotherapy groups. The sMRI navigation group showed significantly higher reduction rates in the somatization/anxiety and retardation subscale scores in both the first and second weeks. More details can be found in Figure 3 . Section title: Main effect test of BSI-CV total score and subscale scores Educational score: 4.120975494384766 Domain: biomedical Document type: Study Language: en Normality and variance homogeneity test were performed on the total BSI-CV score and subscale scores of the 3 groups of patients at different time points during treatment, and the results showed that the total BSI-CV score, SI intensity and suicide risk score of the 3 groups of patients all followed normal distribution and variance homogeneity (P >0.05). The baseline BSI-CV scores among the three groups were not significantly different, indicating no statistically significant differences in suicide at baseline among the three groups (p>0.05). The results showed that time and group had significant interaction on BSI-CV total score (F= 2.031, P <0.001)and SI intensity(F= 7.626, P <0.001). It shows that the effect of time on SI intensity with different treatment methods. Therefore, the time effect and the intergroup effect were tested separately for the three groups of patients. Section title: Individual effect test of BSI-CV total score and its subscale scores Educational score: 4.157792091369629 Domain: biomedical Document type: Study Language: en After 2 weeks of treatment, the BSI-CV score and their subscale scores were all decreased in the sMRI navigation group, the 5-cm group, and the pharmacotherapy group compared to baseline. All subscale scores were significantly reduced as the corresponding treatments progressed . There were no significant differences in the total BSI score and its factor scores at baseline among the three groups. The results showed that compared with before treatment, the total BSI-CV score and its subscale scores in the 3 groups were significantly reduced with the corresponding treatment progress. The BSI-CV total and subscale scores decreased significantly over time in all groups: baseline>1week (P <0.001) > 2 weeks (P <0.001). Further, the total score of BSI-CV, SI intensity and suicide risk scores of the three groups were compared at the same time point. There were no significant differences between the three groups in BSI-CV total score, SI intensity and suicide risk at first week. At the end of the second week of treatment, the total score of BSI-CV in sMRI navigation was lower pharmacotherapy alone group(P<0.001), there was no significant difference between the other groups(P=0.023). The SI intensity score of sMRI navigation group was significantly lower than 5-cm positioning group (P<0.001) and pharmacotherapy alone group (P<0.001), and there was no significant difference between 5-cm positioning group and pharmacotherapy alone(p=0.672). There was no significant difference in suicide risk among the three groups(P=0.744), detailed results are shown in Table 3 . Section title: Assessment of adverse reactions in patients of the sMRI navigation group and the 5-cm positioning group after treatment Educational score: 4.179544448852539 Domain: biomedical Document type: Study Language: en When administering rTMS treatment to adolescents with depression, the most common side effects include dizziness and localized headaches, but these symptoms can be alleviated after the treatment concludes. Seizures are the most severe side effect of rTMS, and medication use is a possible cause of seizure induction. Adolescents, as a special group, may have a lower threshold for seizure induction compared to adults, making it crucial to understand the patient’s medication history before conducting rTMS treatment. Additionally, a study pointed out that excessively high stimulation intensity of rTMS can also induce seizures, so timely assessment and adjustment of the stimulation intensity are important measures to ensure the safety of rTMS ( 30 ). During the treatment process, among all adolescent patients diagnosed with MDD, three patients reported dizziness after the first rTMS treatment, with an incidence rate of 2.4%. The dizziness they experienced was mild and transient, resolving after a short rest, and did not recur in subsequent treatments. The remaining patients did not report any significant adverse reactions, and no seizures occurred, indicating a general tolerance and acceptance of the treatment. Section title: Discussion Educational score: 4.1028900146484375 Domain: biomedical Document type: Study Language: en This study of adolescent MDD patients utilized sMRI-guided rTMS for precise positioning, as well as multidimensional assessment of the emotional state and SI before and after rTMS treatment. A pharmacotherapy group served as the control. The sMRI navigation group showed significant and rapid anti-depressant efficacy, as well as reduced SI. Moreover, adolescent MDD patients showed high tolerance to 10Hz sMRI-navigated rTMS. No severe adverse reactions such as serious headaches, pain at the stimulation site, or epilepsy were encountered during the treatment period, thus demonstrating the safety and tolerability of 10Hz sMRI-guided rTMS treatment in MDD adolescents. In summary, 10Hz sMRI-guided rTMS is a safe, effective, and feasible anti-depressant treatment. This opens a new strategy for the clinical treatment of adolescent MDD. Section title: Discussion Educational score: 4.1119704246521 Domain: biomedical Document type: Study Language: en Analysis of the changes in HAMD-17 scores during treatment revealed a significant interaction between treatment time and group. This suggests the improvement of clinical symptoms in the different treatment groups varied with time. After the first and second weeks of treatment, The sMRI navigation group showed lower scores and greater efficacy than other groups, the rTMS combined with pharmacotherapy groups had significantly lower HAMD scores than the pharmacotherapy alone group. Section title: Discussion Educational score: 4.29286527633667 Domain: biomedical Document type: Study Language: en This suggests that rTMS is effective at stimulating the rapid onset of action of anti-depressant drugs. The standard rTMS protocol approved by the FDA for treating adult depression targets the L-DLPFC with 10Hz stimulation ( 31 ). Given that younger age is a favorable factor for the effectiveness of rTMS, researchers have begun to explore the efficacy and safety of rTMS in children and adolescents to develop suitable protocols for these age groups. One study showed that after treatment with 10Hz rTMS, adolescents showed significant improvement in depression symptoms, as indicated by scores on the Children’s Depression Rating Scale-Revised (CDRS-R), and there was also significant improvement in scores on the Clinical Global Impressions-Severity scale (CGI-S) ( 32 ). The rTMS treatment conducted by Croarkin on 10 adolescents with treatment-resistant depression showed an increase in the glutamate/glutamine ratio after treatment, which was negatively correlated with the severity of depression. This suggests that rTMS may improve depressive symptoms by modulating glutamate neurotransmission ( 33 ). MRI study also found that rTMS can induce changes in the amplitude of low-frequency fluctuations (ALFF), regional homogeneity (ReHo), and functional connectivity strength (FCS) in brain regions associated with depression ( 34 ). Research on adults indicates that treatment duration, specifically 10 rTMS sessions within two weeks, was effective at improving depressive symptoms, highlighting the importance of treatment duration for TMS efficacy ( 35 ). Consistent with this, the current study also found that minors showed significant improvement in depressive symptoms after 10 rTMS sessions. The combination of rTMS with anti-depressant medication presents notable advantages due to the synergistic effects. Medication improves mood by regulating neurotransmitter levels in the brain, while rTMS exerts therapeutic effects by non-invasively stimulating neural activity in specific brain areas. When used together, rTMS can target brain areas that are otherwise challenging for medication alone, thus providing a more comprehensive treatment approach. The sMRI navigation and 5-cm positioning groups showed higher rates of reduction in the anxiety/somatization scores than the pharmacotherapy group. These scores included depressive mood, loss of interest, anxiety, and somatic anxiety, reflecting the core symptoms of MDD. The combination of rTMS with medication may therefore offer notable benefits in improving depressive symptoms through additive effects, especially by improving retardation and anxiety and enhancing interest in activities. Section title: Discussion Educational score: 4.096452236175537 Domain: biomedical Document type: Study Language: en This study also explored whether rTMS treatment improves SI in MDD adolescents. SI in all three groups improved after treatment, consistent with previous research findings ( 36 – 39 ). Multifactorial repeated measures ANOVA revealed a significant interaction between treatment time and group during the treatment period. This finding suggests differences between the three treatment groups in terms of SI changes. After the second week of treatment, the sMRI navigation group showed significantly lower total BSI-CV scores and SI severity compared to the 5-cm positioning and pharmacotherapy groups. A one-week double-blind study found that neuro navigation-guided high-dose rTMS significantly improved depressive symptoms and reduced suicidal ideation in MDD patients ( 40 ). Section title: Discussion Educational score: 4.283525466918945 Domain: biomedical Document type: Study Language: en Furthermore, the SI of patients in the sMRI navigation group decreased notably after the first week of treatment compared to sham stimulation. Additionally, 3 days of high-frequency rTMS on the DLPFC area significantly reduced SI in adult MDD patients ( 41 ). The rapid onset of action could be due to the thrice daily 30-minute sessions of high-frequency rTMS, resulting in a total of 54,000 stimuli. rTMS targets the DLPFC to enhance neural activity in this area, thereby improving emotion regulation, cognitive control, and impulse inhibition, which are crucial for reducing suicidal ideation. Currently, adolescents typically do not undergo such high-frequency and high-intensity stimulation protocols due to safety considerations. RTMS can reshape the connectivity of key neural networks in the brain, including the default mode network (DMN) and the central executive network (CEN), optimizing brain functional integration and enhancing emotional and cognitive processing. Neurotransmitter modulation is also an important mechanism of rTMS; by affecting levels of neurotransmitters such as dopamine and glutamate, rTMS provides a biological basis for improving depressive symptoms and reducing suicidal ideation ( 42 ). More importantly, rTMS promotes neuroplasticity through repetitive stimulation, leading to adaptive structural and functional changes in the brain. This helps establish healthier emotional and behavioral patterns and enhances patients’ psychological resilience. The combined effects of these mechanisms provide a solid theoretical foundation and empirical support for the use of rTMS in intervening in adolescent suicidal ideation ( 26 ).Currently, there is no consensus on the feasibility and safety of multiple daily sessions of rTMS in the adolescent population. Due to safety considerations, we did not apply multiple daily rTMS for our adolescent patients. This exploratory study in adolescents found that SI decreased significantly after 10 sessions of rTMS over 2 weeks. Anti-depressant medications typically take effect after 2 weeks. The current study revealed that rTMS combined with medication can effectively control early-stage SI and improve clinical symptoms after 2 weeks, leading to a substantial reduction in early suicide risk. This suggests that the combined treatment model provides prompt relief of SI in patients, with rapid onset and a high safety profile. SI is one of the strongest predictors of suicidal behavior and usually precedes suicide intention and behavior ( 43 , 44 ). Active and effective intervention is particularly critical in ensuring the life safety and quality of life of MDD patients. The present study not only provides evidence for the safety of rTMS treatment in adolescents, but also suggests that rTMS is a potential treatment option for reducing SI. Section title: Discussion Educational score: 4.205567836761475 Domain: biomedical Document type: Study Language: en Overall, the sMRI navigation group showed a greater reduction of depressive symptoms and SI than both the surface 5-cm positioning and pharmacotherapy groups. Precision localization of TMS, such as with the use of a neuro-navigation system, is superior to traditional surface localization methods because it allows individualized localization based on sMRI. The combination of sMRI and neuro-navigation for TMS localization enables the reconstruction of head models using participant-specific sMRI data. Compared to the traditional 5-cm positioning method, this results in a better match of the real head shape of the participant ( 24 ), thereby reducing heterogeneity due to differences in individual anatomical structures. Precise localization can reduce stimulation of surrounding non-target areas, enhance the efficacy and response rate of rTMS, and thus minimize any side effects. Personalized rTMS/TBS protocols were incorporated with neuroimaging in a retrospective analysis of the therapeutic effect of individualized rTMS for depression ( 45 ). By acquiring three-dimensional (3D) T1-weighted structural MRI data from subjects and inputting it into a neuronavigation system, brain structures can be visualized, which has a positive impact on improving the accuracy of target localization. Fitzgerald ( 46 ) conducted a randomized controlled trial involving 51 patients with treatment-resistant depression, who were randomly divided into two groups: the “5 cm localization method” group and the neuronavigation group The results showed that the neuronavigation group had better outcomes. This demonstrated the benefits of personalized stimulation parameters for improving clinical outcomes. Such methods require further validation in future clinical trials. During the treatment process, no severe adverse events including severe headaches or epilepsy were observed ( 47 ), and no related adverse reactions such as flashes of light, blurred vision, or hallucinations occurred. Indeed, only three patients in the present study experienced dizziness after treatment. This was alleviated after rest, indicating relatively good tolerability and few major side effects from the treatment. Section title: Discussion Educational score: 3.8693230152130127 Domain: biomedical Document type: Study Language: en As previously mentioned, the enrolled participants in our study were complicated with other disorders. They were not excluded, however, because their comorbidities reflect the real-world conditions of MDD adolescents ( 48 ). Although these comorbid diseases were not assessed here, previous studies have reported on the therapeutic effects of rTMS for obsessive-compulsive disorder and anxiety disorders ( 49 ). However, larger study cohorts are required to allow more comprehensive assessments. The risk of adolescents with MDD transitioning to bipolar disorder (BD) is estimated at 45% ( 50 ). To address this, we applied strict inclusion criteria, assessing family history and manic symptoms to minimize high-risk cases. We also plan long-term follow-up to monitor BD emergence, identify predictors. Section title: Discussion Educational score: 4.10544490814209 Domain: biomedical Document type: Study Language: en In this study, the sMRI navigation group showed favorable anti-depressant efficacy and reduction of SI compared to the 5-cm positioning and pharmacotherapy groups. This combined approach may be an important way to further enhance the efficacy of treatment for MDD in adolescents. However, this study also had several limitations, including the relatively small sample size. Although information was available for the duration of illness at baseline, there was a lack of data on the number of episodes and the specific medication. Therefore, the current results require confirmation by performing multi-center, randomized controlled trials with a larger sample size. Considering the heterogeneity of MDD, further investigation of different MDD subtypes is also required in order to achieve individualized and precise rTMS intervention strategies. This is necessary for both clinical symptom and biological subtypes, with the goal of providing adequate evidence for the application of rTMS in the treatment of depressive disorders. Additionally, we did not set up sham control treatment after patient enrollment. Anti-suicidal treatment in adolescents remains challenging. In particular, determining the capacity of adolescents to consent to research intervention is a complex process in the context of emotional dysregulation, historically suboptimal decision-making, and recent suicide attempts. Several limitations must be considered in the current study. Moreover, as part of the study design, participants were required to use only one type of anti-depressant medication during treatment and not change the type of medication. Although we inferred the observed clinical changes as being attributable to rTMS rather than to previous treatments, we cannot exclude a possible impact of previous treatments on the clinical outcomes. Section title: Conclusion Educational score: 4.1079912185668945 Domain: biomedical Document type: Study Language: en This study employed an open-label design and combined two weeks of high-frequency rTMS treatment with anti-depressant medication. sMRI-guided rTMS treatment was found to be more effective than the traditional 5-cm positioning method and pharmacotherapy. Therefore, 10Hz rTMS based on individualized and precise sMRI of the DLPFC region can improve the clinical symptoms and SI of adolescents with MDD. This combined approach may be a feasible, effective, and safe treatment option for such patients. | Review | biomedical | en | 0.999998 |
PMC11695404 | Section title: Introduction Educational score: 4.056264877319336 Domain: biomedical Document type: Review Language: en Stroke, clinically referred to as cerebrovascular accident, is a syndrome characterized by brain dysfunction resulting from pathological events in the brain’s blood vessels such as arterial embolism, small vessel disease, or cerebral infarction. Clinically, it primarily manifests as two types: hemorrhagic and ischemic, with the latter being more predominant in terms of incidence . Global burden of disease studies have shown that stroke is one of the leading causes of death and disability worldwide, posing a significant economic burden . With the aging population, the prevalence of stroke is expected to rise in the coming decades, exacerbating economic and social pressures. Numerous risk factors contribute to stroke, including but not limited to age, sex, genetic predisposition, and lifestyle factors, among which chronic diseases and unhealthy habits are recognized as critical risk factors . Section title: Introduction Educational score: 4.298920631408691 Domain: biomedical Document type: Study Language: en Joint replacement surgery, particularly hip and knee replacements, has become an established effective method for treating advanced joint pain and functional impairment . Given the global trend of population aging, it is projected that by 2040, the number of joint replacement surgeries performed annually in the United States will approach 5 million . While joint replacement surgeries provide significant symptom relief and functional recovery, postoperative complications such as neurovascular injuries, deep vein thrombosis (DVT), and prosthetic infections should not be overlooked . Furthermore, clinical studies have indicated that patients undergoing total hip replacement face a heightened risk of stroke . Joint prostheses are often made from metal alloys, including cobalt-chromium alloys, stainless steel, titanium alloys, as well as biocompatible materials like ceramics and polymers . These metal implants endure mechanical stress and the body’s fluid environment (such as blood, tissue fluid, and lymphatic fluid) over the long term, potentially leading to localized and systemic release of metal ions . Components in the body fluid environment, such as electrolytes (such as sodium ions and chloride ions), proteins, and other organic molecules, can affect the corrosion behavior of the metal surface through chemical and electrochemical reactions, leading to the release of metal ions. Notably, cobalt has been shown to have potential cardiotoxic effects, with significant increases in cobalt concentrations in myocardial cells following total hip replacement . Section title: Introduction Educational score: 4.030620098114014 Domain: biomedical Document type: Study Language: en Based on this background, the present cross-sectional study aims to utilize data from the National Health and Nutrition Examination Survey (NHANES) to explore the relationship between metal implants and stroke risk among U.S. adults. We hypothesize that individuals with metal implants have a higher risk of stroke compared to those without such implants. The findings of this study are expected to provide new scientific evidence regarding the association between metal implants and stroke risk, offering theoretical support for clinical decision-making, patient counseling, and public health policy formulation. Section title: Study population Educational score: 4.04055118560791 Domain: biomedical Document type: Study Language: en The National Health and Nutrition Examination Survey (NHANES) is a nationally representative survey conducted across the United States, employing random sampling to select participants, ensuring that the data is representative of the entire U.S. population. Implemented biennially, each cycle of the survey encompasses face-to-face interviews, physical examinations, laboratory tests, and other health measurements for thousands of residents. The data from NHANES are extensively utilized for formulating public health policies, guiding research, and evaluating national health objectives. It should be noted that, while the NHANES data provide nationally representative information, the study was unable to analyze potential regional differences due to the inability to obtain specific geographic area data. This may limit the extrapolation of the results to certain specific areas. However, this limitation does not detract from the national applicability of the results, as the sampling method of NHANES ensures broad representativeness of the data. Future studies can combine other databases with geographic area details for further exploration. A total of 37,464 participants were obtained in the selected NHANES cycle. We further excluded subjects with missing questionnaire data and relevant laboratory tests. Ultimately, a total of 12,337 participants were included in this study. Figure 1 illustrates the flowchart for participant selection. Section title: Measurement and definition Educational score: 3.9874041080474854 Domain: biomedical Document type: Study Language: en The presence of metal implants in the body was determined based on an affirmative response to the following question: “Do you have any artificial joints, pins, plates, metal sutures, or other types of metal objects in your body?” Additionally, the mentioned metal implants do not include piercings, dental crowns, braces or fixtures, shrapnel, or bullets, and should not be visible externally or in the mouth. Stroke diagnosis was further assessed using a health questionnaire. During the interview, participants were asked, “Have you ever been diagnosed with a stroke by a doctor or other healthcare professional?” Participants who answered “yes” to this question were considered stroke survivors. This definition has been used in numerous epidemiological studies and has been effective in capturing health events among participants. However, due to the lack of radiological confirmation or medical records, future studies should employ a variety of methods to verify the accuracy of stroke diagnoses. Section title: Covariates Educational score: 4.087839126586914 Domain: biomedical Document type: Study Language: en In line with previous studies, we collected variables that may be associated with the relationship between metallic implants and stroke, including smoking, alcohol consumption, obesity, and diabetes. We obtained sociodemographic data (such as gender, age, race/ethnicity, poverty status [PIR], education level, and body mass index [BMI]), lifestyle factors (such as smoking and drinking status), and medical history (such as diabetes) as covariates . Education levels were categorized into three groups used by NHANES: high school or below, high school equivalent, and college or above. Participants were classified as Mexican American, other Hispanic, Hispanic white, non-Hispanic black, or other races. The PIR was divided into three categories: low (<1.5), medium (1.5–3.5), and high (>3.5). Marital status was defined as one of three groups: married and living with a partner, other (including widowed, divorced, or separated), and never married. For lifestyle factors, individuals who reported having smoked more than 100 cigarettes in their lifetime were classified as smokers. Alcohol consumption was assessed through the questions: “During the past year, have you consumed at least 12 drinks of any kind of alcoholic beverage? By “one drink,” I mean a 12-ounce beer, a 5-ounce glass of wine, or a 1.5-ounce shot of spirits?” . Diabetes was defined as self-reported diabetes, use of antidiabetic medications, or meeting the criteria set by the American Diabetes Association (ADA). Participants were classified as having hypertension if they met any of the following criteria: average systolic blood pressure (SBP) ≥ 140 mmHg average diastolic blood pressure (DBP) ≥ 90 mmHg self-reported diagnosis of hypertension; or currently using antihypertensive medications. Coronary heart disease was determined based on a questionnaire asking participants whether a doctor had ever informed them of this condition. Section title: Statistical analysis Educational score: 3.4140918254852295 Domain: biomedical Document type: Study Language: en Statistical analyses were conducted using Stata 16 and R software version 4.2.1. Descriptive statistics were employed to present the data, with normally distributed or skewed data reported as mean ± standard deviation, and categorical variables expressed as counts and percentages. Complex sample weights were utilized to estimate population characteristics, BMI, and the overall prevalence of diabetes and hypertension. Section title: Statistical analysis Educational score: 4.061248779296875 Domain: biomedical Document type: Study Language: en Subsequently, statistically significant covariates were incorporated into a multiple linear regression model for analysis. In this study, metal implants served as the predictor variable, while stroke was the outcome variable for the association study. After adjusting for covariates, the relationship between metal implants and stroke was further explored, and effect sizes ( β ) along with their 95% confidence intervals (CI) were calculated. To enhance the accuracy of the results, subgroup analyses stratified by age, gender, and physical activity were performed. A p -value of <0.05 was considered statistically significant for assessing the performance of the metrics. Section title: Population-based research analysis Educational score: 3.9201266765594482 Domain: biomedical Document type: Study Language: en A total of 12,337 eligible participants were included in this study, comprising a metal implant group ( n = 3,699) and a non-metal implant group ( n = 8,638). The average age of participants was 58.570 years (standard error ± 11.565), with 47.624% being male. Among them, 753 individuals had a history of stroke. Table 1 presents the demographic and baseline characteristics of the metal implant and non-metal implant groups. Notably, the metal implant group was significantly older than the non-metal implant group ( p < 0.001). The proportion of non-Hispanic whites in the metal implant group was higher than in the non-metal implant group (75.621 vs. 63.940%). Section title: Population-based research analysis Educational score: 3.950176954269409 Domain: biomedical Document type: Study Language: en Furthermore, the metal implant group exhibited higher rates of poverty, lower education levels, smoking, alcohol consumption, BMI, hypertension, coronary heart disease, physical inactivity, and diabetes (all p < 0.05). Importantly, the prevalence of stroke was higher in the metal implant group compared to the non-metal implant group (7.395 vs. 3.597%, p < 0.001). Section title: Population-based research analysis Educational score: 4.096961975097656 Domain: biomedical Document type: Study Language: en Table 2 presents the logistic regression analysis with metal implants as the independent variable and stroke as the outcome. Overall, metal implants were significantly associated with stroke [OR = 2.140, 95% CI (1.720, 2.663), p < 0.001]. This positive correlation persisted even after adjusting for other variables, remaining significant in Model 2 [OR = 1.835, 95% CI (1.463, 2.301)], Model 3 (OR = 1.650, 95% CI (1.305, 2.088), p < 0.001), and Model 4 (OR = 1.458, 95% CI (1.130, 1.881), p = 0.004). In all models, the presence of metal implants was positively correlated with the incidence of stroke, and this association remained statistically significant. Section title: Population-based research analysis Educational score: 4.065812110900879 Domain: biomedical Document type: Study Language: en Table 3 illustrates the results of the stratified analysis, which demonstrates that the correlation between metal implants and stroke increases with age and decreases in physical activity. The relationships in the final model are as follows: for the >60 age group [OR = 1.513, 95% CI (1.129, 2.028), p = 0.006], for the group with low physical activity [OR = 1.382, 95% CI (1.043, 1.829), p = 0.024], and for women [OR = 1.382, 95% CI (1.043, 1.829), p = 0.024]. Section title: Discussion Educational score: 4.097245216369629 Domain: biomedical Document type: Study Language: en This study utilized nationally representative data from the NHANES to elucidate the strong association between metal implants and stroke. Importantly, this association remained statistically significant even after adjusting for several confounding factors, including age, gender, race, physical activity, socioeconomic status, obesity, smoking status, alcohol consumption, hypertension, and diabetes. In our research, approximately 7.395% of patients in the metal implant group had a history of stroke, which is 3.798% higher than that in the non-metal implant group. These findings prompt us to consider the possibility that metal implants may be a potential risk factor for stroke. In subgroup analysis, it has been observed that women who undergo metal implantation are at an increased risk of stroke compared to their male counterparts. Research indicates that women tend to have higher concentrations of metal ions in their bodies . Furthermore, individuals over the age of 60 are at a higher risk of developing stroke, and those with less physical activity also face an elevated risk. Studies have shown that the proportion of non-Hispanic whites in the group with metal implants is significantly higher than that of other ethnicities, which may reflect differences in access to medical resources, surgical preferences, and cultural factors among different ethnic groups. For instance, research has indicated that non-Hispanic whites are more inclined to undergo joint replacement surgery or other treatment measures involving metal implants . This racial distribution difference may suggest that racial factors play a significant role in the association between metal implants and stroke risk. Therefore, this distribution characteristic emphasizes the importance of balancing racial representation in future study designs. Section title: Discussion Educational score: 4.673910140991211 Domain: biomedical Document type: Study Language: en Previous studies have primarily focused on the release of metal ions due to wear or corrosion of metal implants. For instance, some researchers have identified mechanical-assisted crevice corrosion (MACC) following total hip arthroplasty, during which metal ions may enter the bloodstream . The accumulation of these ions in the body can be disseminated through circulation, potentially affecting overall health, including vascular health . Although there is currently no direct evidence linking metal ions to stroke causation, studies have indicated that the normal functioning of brain tissue relies on essential ion transporters. The accumulation of ions may disrupt the operation of these transporters, potentially leading to a vicious cycle that increases stroke risk . Additionally, research has shown that cobalt ions can lead to apoptosis and necrosis of endothelial cells once they diffuse into the bloodstream, thereby inhibiting the activity of other cells and increasing stroke risk . The potential effects of metal ions on cardiac function, including disruption of heart rhythms and impairment of valve function, may also indirectly elevate the risk of stroke. One study observed that cobalt ions exhibited cytotoxic effects on synovial cells (fibroblasts), causing inflammation in hips with metal implants . Furthermore, cobalt ions can affect mitochondrial function, leading to autophagy of mitochondria and triggering hypoxic responses . Hypoxic responses in cells include the secretion of cytokines that can provoke inflammation by activating vasculature and enhancing leukocyte migration . Metal ions may also have widespread effects on biological systems by inducing oxidative stress responses. For instance, studies have shown that titanium dioxide nanoparticles can trigger oxidative stress, leading to repeated cellular damage and abnormal proliferation, while also causing indirect DNA damage . These processes may lead to abnormal cell growth behavior and further promote osteolysis around the prosthesis . In addition, some studies have found that increased titanium concentrations in plasma are significantly associated with an increased risk of stroke and heart disease . Section title: Discussion Educational score: 4.061614036560059 Domain: biomedical Document type: Study Language: en Furthermore, metal allergic reactions are also a concern following joint replacement surgery. Some patients may develop allergic reactions to certain components in metal implants, such as cobalt and chromium, potentially leading to local tissue reactions or even systemic responses. In a study by Bloemke and Clarke , a self-reported survey on skin allergies, metal allergies, or sensitivities was conducted among patients undergoing primary total knee arthroplasty (TKA), revealing that individuals with these allergic symptoms comprised as much as 14% of the sample. In contrast, Nam et al. found a self-reported incidence of metal allergies at 4.1% through analysis of a large cohort . Additionally, the immune response triggered by metal allergies may indirectly affect cardiac function, increasing the risk of arrhythmias and changes in blood composition, all of which could promote the occurrence of strokes . Therefore, although direct evidence is lacking, the potential link between metal allergies and stroke should not be overlooked, necessitating further research to explore these underlying mechanisms and provide patients with more comprehensive prevention and treatment strategies. Section title: Discussion Educational score: 4.344614028930664 Domain: biomedical Document type: Review Language: en Research indicates that levels of physical activity may play a significant modulating role between metal implants and the risk of stroke. Particularly in groups with low physical activity, the positive correlation between metal implants and stroke risk is more pronounced, which may be related to the hemodynamic changes and decreased metabolic levels triggered by insufficient physical activity . Furthermore, studies have shown that replacing 1 h of sedentary time (ST) with an equivalent amount of physical activity (PA) per week can significantly reduce the risk of all-cause mortality, cardiovascular disease, and cancer deaths . Metal ions in the body can promote vascular pathology by activating inflammatory factors. However, moderate physical activity can not only neutralize the potential side effects of metal ions but also improve overall prognosis by raising the patient’s health threshold . In contrast, although high-intensity physical activity may increase joint load and exacerbate implant wear, thereby releasing more metal ions, appropriate physical activity helps to balance this risk. It is worth mentioning that a study found that moderate physical activity can also reduce the levels of heavy metal ions such as cadmium and lead in the blood, thereby alleviating their toxic effects on the vascular system and further reducing the potential risk of stroke . This evidence suggests that physical activity not only has a significant impact on the biological effects of implant wear and metal ion release but may also indirectly reduce the risk of stroke by modulating inflammatory responses and improving vascular function. Section title: Discussion Educational score: 3.8145947456359863 Domain: biomedical Document type: Review Language: en In summary, although the direct relationship between metal implants and stroke following joint replacement surgery has yet to be definitively established, the release of metal ions, metal allergic reactions, and the necessity for long-term health monitoring in patients represent key areas for future research. Future studies should explore how these factors collectively impact patients’ long-term health outcomes and their potential influence on stroke risk. Section title: Discussion Educational score: 4.157873630523682 Domain: biomedical Document type: Study Language: en Additionally, the NHANES database lacks detailed records regarding metal implants, including the type, number, duration within the body, and key information such as the corrosion rates and ion release profiles of different metal materials. This data absence limits a comprehensive assessment of the potential impact of different metal types on stroke risk. Moreover, significant differences may exist in functionality, biomechanical load, and in vivo degradation processes of different metal implants, which could influence study outcomes, increase heterogeneity between groups, and reduce the clinical specificity of study conclusions. Lastly, a limitation of this study is the significant difference in racial distribution between the metal implant group and the non-implant group, which may affect the universality of the results. Although we adjusted for racial factors in multivariate regression analysis, this imbalanced distribution may still mask specific associations within certain subgroups. Future research will aim to enhance clinical relevance and specificity by collecting more detailed information regarding metal implants and addressing potential confounding factors. To overcome these limitations, prospective study designs should be employed in future research. Moreover, we must acknowledge the possibility of unmeasured or unknown confounding factors, which could lead to residual confounding effects in the study. | Review | biomedical | en | 0.999999 |
PMC11695405 | Section title: Introduction Educational score: 4.320229530334473 Domain: biomedical Document type: Review Language: en Mesenchymal stem cells (MSCs), which are multipotent progenitor stromal cells, possess the remarkable potential of self-renewal and the ability to differentiate into multiple mesenchymal cell lineages. MSCs were first discovered in bone marrow by Friedenstein et al. and were later named mesenchymal stem cells by Caplan et al. ( 1 – 4 ). Pittenger et al. further discovered that adult stem cells could be induced to specifically differentiate into adipocytes, chondrocytes, or osteoblasts ( 5 ). And subsequent researchers found that MSCs could exist in various tissues such as adipose tissue, umbilical cord, and pulp, further highlighting their importance and potential therapeutic applications ( 6 , 7 ). Besides possessing remarkable self-renewal capabilities, MSCs also exhibit extraordinary potential to differentiate into various cell lineages. One of the most notable features of MSCs is their capacity for self-renewal, enabling them to serve as promising candidates for cell and regenerative therapies ( 8 , 9 ). Furthermore, these cells possess the versatility to differentiate into mesoderm lineages and, under specific conditions, even into ectoderm and endoderm tissues, showcasing their developmental plasticity ( 10 ). Importantly, studies indicate that MSCs exhibit a lower tumorigenesis risk due to their differentiation pathway typically avoiding malignant transformation, thereby alleviating concerns regarding the safety and side effects associated with stem cell therapies ( 11 , 12 ). In recent years, extensive research has demonstrated that MSCs hold significant immunomodulatory potential ( 13 – 15 ). This makes them extremely promising candidates for the therapy of autoimmune inflammatory disorders. The immunomodulatory mechanisms of MSCs are primarily exerted through interactions with immune cells via direct cell contact and paracrine activity ( 16 ). Autoimmune disorders emerge as a result from an imbalance in the immune system that disturbs immunological tolerance ( 17 ). Most patients afflicted with autoimmune disorders require long-term, and in some cases even lifelong, therapies in order to maintain a basic quality of life. Some first-line drugs that primarily target interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) molecules have been shown to be beneficial in clinical treatment ( 18 , 19 ). However, patients who use immunosuppressive drugs for long-term or high dosage may suffer from drug resistance and adverse reactions. These adverse reactions can include an increased risk of infection and the development of malignancy. Therefore, it is urgent to develop comprehensive treatment, combined with MSCs supplementary treatment. By doing so, we can harness the immunomodulatory potential of MSCs and achieve the crucial goal of controlling disease progression. Here, we review the immunomodulatory characteristics of MSCs and their recent applications in autoimmune diseases, so as to provide a basis for the practical application of MSCs. Section title: Immunomodulation of MSCs Educational score: 4.297023773193359 Domain: biomedical Document type: Review Language: en MSCs possess remarkable immunoregulatory properties that make them a subject of great interest in the field of immunology and regenerative medicine. MSCs participate in both innate and adaptive immune responses through direct cell contact and production of paracrine mediators. MSCs can directly interact with various immune cells, such as T cells, B cells, macrophages, natural killer cells (NKs), dendritic cells (DCs) and neutrophils ( 20 , 21 ). Through these direct contacts, MSCs can modulate the activation, proliferation, and differentiation of immune cells. Additionally, MSCs secrete a variety of bioactive molecules, including cytokines, growth factors, and chemokines, which act in a paracrine manner to influence the behavior and function of immune cells. These paracrine factors can suppress the immune response, promote tissue repair, and regulate the inflammatory environment. The integration of direct cell contacts and paracrine activity equips MSCs with a potent immunomodulatory impact, allowing them to play a crucial role in treating autoimmune inflammatory diseases and various immune-related disorders. These include graft-versus-host disease (GVHD) ( 22 – 24 ), systemic lupus erythematosus (SLE) ( 25 – 27 ), psoriasis ( 28 , 29 ), primary Sjögren’s syndrome (pSS) ( 30 – 32 ), type 1 diabetes mellitus (T1DM) ( 33 – 35 ), rheumatoid arthritis (RA) ( 36 – 38 ), among others ( 39 – 41 ). We summarized the mechanism of action of MSCs in the treatment of autoinflammatory diseases as shown in Figure 1 . Section title: Direct cell contacts with immune cells Educational score: 4.5983052253723145 Domain: biomedical Document type: Review Language: en One of the key immunoregulatory properties of MSCs is their ability to modulate the activity of immune cells. MSCs in a way that is dependent on direct cell-to-cell contact. This means that physical interaction between MSCs and immune cells is crucial for the immunomodulatory process. When MSCs come into contact with immune cells, they can exert their regulatory effects in several ways. For instance, they may modulate the activity of T cells. They can suppress the proliferation and activation of effector T cells, while promoting the generation and function of regulatory T cells. This helps to maintain immune balance and prevent excessive immune responses. MSCs can also interact with B cells. They may influence B cell differentiation, antibody production, and survival. By regulating B cell function, MSCs can contribute to the control of humoral immune responses. In addition, MSCs can have an impact on NKs. They may alter the cytotoxic activity and cytokine production of NKs, thereby modulating innate immune responses. Furthermore, MSCs may interact with DCs. They can affect DCs maturation, antigen presentation, and cytokine secretion. This can lead to changes in the initiation and regulation of adaptive immune responses. Overall, the direct cell contact-dependent mechanisms by which MSCs play an immunomodulatory function on immune cells are complex and multifaceted. These mechanisms play an important role in maintaining immune homeostasis and have potential applications in the treatment of various immune-mediated diseases. Section title: Immune regulation of T cells by MSCs Educational score: 4.732325553894043 Domain: biomedical Document type: Study Language: en MSCs exhibit the expression of programmed cell death-ligand 1 (PD-L1), which engages with the programmed cell death-1 (PD-1) receptor on activated T cells. This interaction suppresses immunity by counterbalancing the activation signals of T cells. The PD-1/PD-L1 signaling pathway constitutes a vital immune checkpoint mechanism, safeguarding healthy tissues from immune attack, thereby playing a pivotal role in modulating immune responses and maintaining immune homeostasis ( 42 , 43 ). Studies indicate that the expression of PD-1 ligands is upregulated in MSCs pretreated with interferon-gamma (IFN-γ), TNF-α, and interleukin-1 beta (IL-1β). Compared to unstimulated MSCs, the pretreated MSCs exhibit enhanced immunosuppressive effects on activated T cells and promote the differentiation of regulatory T cells (Tregs) in a PD-1-dependent manner. Notably, these effects can be reversed by introducing neutralizing antibodies against PD-L1 to MSCs, both in vitro and in vivo ( 44 ). A detailed analysis of single-cell transcriptomics and proteo-transcriptomics of MSCs isolated from various sources, including adipose tissue (AD), bone marrow (BM), placental villi (PM), and umbilical cord (UC), revealing that downregulation of PD-L1 impacts their immunosuppressive activity, which is regulated through GATA2 ( 45 ). In a NOD mouse model, MSCs mitigated the aggregation of T cells and macrophages in pancreatic islets induced by PD-L1/PD-1 blockade, thereby reducing the incidence of diabetes mellitus ( 46 ). When in direct cell-to-cell contact, allogeneic adipose-derived MSCs (ASCs) transfer active mitochondria and plasma membrane segments to Tregs, augmenting their immunosuppressive activity ( 47 ). Additionally, the transfer of MSCs mitochondria to activated CD4 + T cells results in the suppression of Th1 responses ( 48 ). Section title: Immune regulation of T cells by MSCs Educational score: 4.378458023071289 Domain: biomedical Document type: Study Language: en Overexpression of the C-C motif chemokine ligand 5 (CCL5) receptor C-C chemokine receptor 5 (CCR5) enhances the homing ability of MSCs and optimizes their immunomodulatory effects by decreasing infiltrating T cells and activated microglia, and inhibiting the activation of NLRP3 inflammatory vesicles in a mouse model designed to prevent classical experimental autoimmune uveitis (EAU) ( 49 ). Furthermore, direct and indirect interactions with allogeneic MSCs bolster Tregs’ capacity to accumulate immunosuppressive adenosine and inhibit the proliferation of conventional T cells. One direct exchange between Tregs and MSCs involves the transfer of active mitochondrial and plasma membrane fragments from MSCs to Tregs, a process that is HLA-dependent and correlated with a mismatch load of HLA-C and HLA-DRB1 eplet between Tregs and MSCs donors ( 47 ). Co-culture of MSCs derived from mouse inguinal fat with CD4 + T cells sorted from mouse spleen upregulated FcγRIIb expression on CD4 + T cells. Co-culture with sFgl2-MSCs reduced the proportion of Th17 and Th1 cells, increased the proportion of Tregs, and elevated the levels of phosphorylated SHP2 (p-SHP2) and phosphorylated SMAD2/3 (p-SMAD2/3) ( 50 ). Section title: Immunoregulation of MSCs to other immune cells Educational score: 4.235895156860352 Domain: biomedical Document type: Study Language: en MSCs exert an influence on B cells through cell-dependent contact mechanisms. Research indicates that MSCs treated with IFN-γ inhibit the production of interleukin-10 (IL-10) by activated B cells via the Cox-2 pathway ( 51 ). Furthermore, MSCs arrest the B cell cycle in the G0/G1 phase, thereby inhibiting cell proliferation, by activating the p38 mitogen-activated protein kinase (MAPK) pathway ( 52 ). Another study showed that B cells and MSCs, when in contact, boosted VEGF production in MSCs. This increase then led to higher levels of pAKT and prevented caspase 3-mediated apoptosis in CD19 + B cells ( 53 ). Section title: Immunoregulation of MSCs to other immune cells Educational score: 4.293100833892822 Domain: biomedical Document type: Study Language: en In the context of innate immune responses, proinflammatory macrophages interact with MSCs, leading to an upregulation of CD200 expression on MSCs. This, in turn, facilitates the reprogramming of macrophages towards an anti-inflammatory phenotype through the binding of CD200 to CD200R on the surface of proinflammatory macrophages ( 54 ). Macrophages in ARDS models can enhance their energy metabolism and antibacterial capacity by phagocytosing mitochondria from MSCs, which are transferred through TNT-like structures ( 55 ). Additionally, MSCs can promote the differentiation of macrophages towards the M2 phenotype through PGE2,TSG-6,TGF-β,and so on Furthermore, MSCs promote the differentiation of macrophages into the M2 phenotype through factors like PGE2, TSG-6, and TGF-β ( 54 , 56 , 57 ). Section title: Immunoregulation of MSCs to other immune cells Educational score: 4.361116409301758 Domain: biomedical Document type: Study Language: en Engineered MSCs with EACAM1 reduce NK cell activation and cytotoxicity through cell-to-cell interactions, regardless of NKG2D ligand regulation. MSCs can also inhibit T lymphocyte and NK cell function via HLA-G5 ( 58 ). Furthermore, MSCs can inhibit the differentiation of DCs through IL-6 ( 59 ). MSCs also release tumor necrosis factor-stimulated gene-6 protein (TSG-6) to suppress the expression of maturation markers on the surface of bone marrow-derived DCs, and reduce the ability of DCs to produce IL-12 and activate T cells ( 59 ). These engineered MSCs also effectively inhibit the proliferation of and activation T cells, as well as the inflammatory responses of monocytes ( 60 ). MSCs can temporarily reside in the lungs and are rapidly phagocytosed by monocytes, subsequently migrating to other parts of the body. After phagocytosing UCMSCs, monocytes polarize towards a non-classical phenotype, expressing PD-L1 and IL-10, while TNF-α levels decrease ( 61 ). Section title: Function in paracrine activity Educational score: 4.069771766662598 Domain: biomedical Document type: Study Language: en Considerable studies have shown that cytokines and exosomes secreted by MSCs can exert their actions on various immune cells via paracrine mechanisms to generate immunomodulatory effects, both in innate and adaptive immune responses. MSCs can also function independently of their cells themselves, but through the bioactive substances they secrete. MSCs-derived extracellular vesicles (MSC-EVs) not only have the same efficacy as MSCs ( 62 , 63 ), but also have some advantages over parental fine MSCs due to their specific miRNA loading ( 64 – 69 ). MSC-EVs is not only easier to preserve, transfer, and produce, but also safer to administer, so it is receiving increasing attention ( 70 ). Section title: Immune regulation of T cells by MSC-EVs Educational score: 4.362529754638672 Domain: biomedical Document type: Study Language: en Recently, research on the modulation of adaptive immunity by MSCs has primarily concentrated on their mechanisms of action on T cell populations, including the regulation of T cell proliferation and the differentiation of Th17 and Treg cells. One study revealed that in vivo administration of MSC-derived extracellular vesicles (MSC-EVs) significantly suppressed CD8 + IFN-γ + cytotoxic T cells (Tc1) and CD4 + IFN-γ + helper T cells (Th1), while decreasing the levels of pro-inflammatory cytokines TNF-α and IFN-γ. Additionally, it induced the generation of CD4 + CD25 + Foxp3 + Tregs and increased the levels of anti-inflammatory IL-10. In vitro experiments from the same study demonstrated that MSC-EVs also inhibited Tc1 and Th1 cells and promoted the induction of Tregs and related cytokines ( 71 ). In dental pulp MSCs (DPMSCs), amino acid deprivation (in humans) and nitric oxide (NO) production (in mice) have been shown to inhibit T cell proliferation within a specific concentration range. However, this inhibitory effect is restricted to local environments due to factors such as the short half-life of NO and the requirement for complete amino acid deprivation ( 72 ). Section title: Immune regulation of T cells by MSC-EVs Educational score: 4.306872367858887 Domain: biomedical Document type: Study Language: en The lactate-dependent immunosuppressor mechanism was confirmed in UC-MSCs through the (delayed-type hypersensitivity, DTH) inflammatory model, and inhibition of lactate dehydrogenase (LDH) significantly reduced the ability of UC-MSCs to control the proliferation of activated CD4 + and CD8 + human T cells. Furthermore, high concentrations of L-lactic acid (25-50 mM) were found to profoundly affect the proliferation and differentiation of Th1 and Th17 cells ( 73 ). Research has demonstrated that exosomes derived from human umbilical cord mesenchymal stem cells (UCMSC-Exos) exhibit inhibitory effects on the abnormal proliferation and apoptosis of CD4 + T cells in individuals with pSS. These exosomes impede the G0/G1 cell cycle phase, prevent the progression into the S phase, diminish Th17 cell differentiation, and foster the differentiation of Tregs. Furthermore, UCMSC-Exos suppress the secretion of cytokines such as IFN-γ, TNF-α, IL-6, interleukin-17A (IL-17A), and IL-17F, while enhancing the production of IL-10 and TGF-β in pSS patients ( 74 ). Section title: Immune regulation of T cells by MSC-EVs Educational score: 4.579133987426758 Domain: biomedical Document type: Study Language: en When Fas mutant mice were systemically supplemented with apoptotic vesicles (ApoVs), which directly interacted with CD4 + T cells, they inhibited CD25 expression and IL-2 production in a dose-dependent manner. The phosphatidylserine exposed on ApoVs mediated interactions with T cells, disrupting proximal T cell receptor signaling, reducing Th17 differentiation and memory T cell formation, and ameliorating inflammation and joint erosion in murine arthritis ( 75 ). Chen et al. prepared exosomes derived from human placental mesenchymal stem cells (hpMSC-ExoMSC), and RNAseq analysis demonstrated that Th17 differentiation was inhibited. Furthermore, hpMSC-ExoMSC improved the hypersecretory phenotype and cell-cell interactions in the hepatic Th17 microenvironment by modulating PERK/CHOP signaling ( 76 ). Using a mouse model of experimental autoimmune uveitis (EAU) and other in vitro assays, Kaur et al. demonstrated that MSC-EVs inhibit the MAPK/ERK pathway in activated T cells. Treatment with TGF-β1 or let-7b had a similar effect on the MAPK/ERK pathway ( 77 ). Cheung et al. analyzed the transcriptome of apoptotic MSCs and found that the ApoMSC secretome-dependent cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) axis inhibited the proliferation and activation of human T cells and exerted chemo attractive effects on monocytes in vitro . Caspase activation under the regulation of the nuclear factor-kappa B (NF-κB) pathway induced upregulation of a range of immunosuppressive molecules in apoptotic MSCs, including COX2/PGE2, CCL2, LIF, TSG-6, and IL-6 ( 78 ). Ni et al. revealed that high expression of the CCL21-CCR7 axis in mice with autoimmune diseases promotes the targeted homing of MSCs to lymph nodes, thereby driving the specific distribution of MSCs. The results suggest that elevated levels of TNF-α, via the NF-κB pathway, induced transplanted MSCs to stimulate the secretion of L-amino acid oxidase (LAAO), which significantly inhibited Th17 cells. Additionally, indole-3-pyruvate (I3P) derived from LAAO acts as an effective inhibitor of Th17 cells by activating the aryl hydrocarbon receptor (AHR) pathway ( 79 ). Section title: Immunomodulatory effects of MSC-EVs on B cells Educational score: 4.595259666442871 Domain: biomedical Document type: Study Language: en Currently, the precise mechanism by which MSCs regulate B cell immunity remains elusive. However, numerous in vivo and in vitro studies have revealed that MSCs can inhibit B cell proliferation and differentiation to a certain degree. Research conducted in CCL2 KO mice demonstrated that the absence of CCL2 augments B-cell receptor (BCR) signaling by upregulating the phosphorylation of the MST1-mTORC1-STAT1 axis. This leads to a reduction in marginal zone (MZ) B cells and an increase in germinal center (GC) B cells. In vivo inhibition of mTORC1 reverses the abnormal differentiation of MZ and GC B cells, and specific inhibitors in vitro can also rescue BCR signaling under antigenic stimulation. Additionally, the study found that CCL2 deficiency enhances early B-cell activation, including B-cell migration, clustering, and recruitment of signaling factors, via upregulation of the DOCK8-WASP-actin axis ( 80 ). Furthermore, another study involving lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse models suggested that MSCs may exert therapeutic effects on ALI by reducing the expression of chemokines associated with neutrophil recruitment and immunoglobulin production by lung B cells ( 81 ). In a separate study, olfactory extra mesenchymal stem cell-derived exosomes (Exos@SFMA) effectively alleviated synovial inflammation and joint destruction by significantly decreasing T follicular helper cell responses and further inhibiting GC B cell differentiation into plasma cells ( 39 ). Single-cell RNA sequencing has revealed that MSCs inhibit intrahepatic B-cell proliferation and cytokine production through exosomes, and regulate mitogen-activated protein kinase (MAPK) and nuclear NF-κB signaling pathways. These findings suggest that MSCs and their derived exosomes have diverse and promising therapeutic potential in modulating B cell immunity and related diseases ( 82 ). Section title: MSCs promote the polarization of macrophages towards M2 Educational score: 4.563581466674805 Domain: biomedical Document type: Study Language: en MSCs possess the ability to regulate the activity and polarization of macrophages, thereby exerting immunosuppressive effects. In a LPS-induced abortion model, MSCs were found to promote the transition of decidual macrophages to the M2 phenotype in a manner dependent on tumor necrosis factor-stimulated gene-6 (TSG-6) ( 54 ). The research conducted by Zhuang et al. suggests that TSG-6 plays a pivotal role in regulating the immunomodulatory efficacy of MSCs’ mechanical response, which is modulated by the MAPK and Hippo signaling pathways and the downstream AP1 complex. This, in turn, influences macrophages through the CD44 receptor and inhibits the NF-κB pathway ( 83 ). Investigators discovered that MSCs primed with a TLR5 agonist (KMRC011) increased the secretion of immunosuppressive cytokines such as indoleamine 2,3-dioxygenase (IDO) and COX2, as well as the expression of M2 macrophage-polarizing cytokines like macrophage colony-stimulating factor (M-CSF) and IL-10 in vitro . These findings indicate enhanced immunosuppressive effects on lymphocyte proliferation. Furthermore, in vivo experiments demonstrated that KMRC011-induced MSCs ameliorated the severity of GVHD in a mouse model, with macrophages obtained from the spleens of mice showing a significant increase in the anti-inflammatory M2 phenotype ( 84 ). In a T1DM mouse model, hexyl 5-aminolevulinate (HAL)-loaded engineered cytokine-primed MSCs (H@TI-ev) exhibited high therapeutic efficacy by reducing CD4 + T cell density and activation through the PD-L1/PD-1 axis, and inducing macrophage transformation from M1 to M2 to remodel the immune microenvironment ( 85 ). Moreover, An et al. proposed a therapeutic strategy based on MSCs spheroids. In this strategy, local delivery of encapsulated MSCs spheroids in vivo in rats with myocardial infarction significantly attenuated local inflammation and subsequent fibrosis, while improving cardiac function by mediating the polarization of macrophages toward a healing-promoting M2 phenotype ( 86 ). These findings highlight the diverse and promising therapeutic potential of MSCs and their derivatives in modulating macrophage polarization and related diseases. Section title: MSCs inhibit macrophages Educational score: 4.339118957519531 Domain: biomedical Document type: Study Language: en In recent years, an intriguing hypothesis has emerged among researchers, suggesting that lactate production serves as a crucial mediator of the immunosuppressive activity exhibited by MSCs ( 73 ). A separate study further illuminated this concept by demonstrating that MSCs, both in vitro and in vivo , effectively reduced the expression of CARD9, thereby inhibiting the phosphorylation of NF-κB in macrophages and preventing their polarization into the M1 phenotype ( 87 ). Moreover, adipose-derived stem cells (ASCs) have been shown to secrete STC-1, which negatively regulates the NLRC4 inflammasome in macrophages. This regulatory mechanism contributes to alleviating inflammation in lung tissue during pseudomonas aeruginosa (PA) infection ( 88 ). Additionally, allogeneic ASCs have been proven to play a protective role by enhancing macrophage recruitment and inducing their transition to the M2 phenotype through the HIF-1α/IL-10 signaling pathway ( 89 ). These findings collectively underscore the significant and multifaceted roles of MSCs and ASCs in modulating macrophage behavior and inflammation. Section title: MSCs interact with macrophages Educational score: 4.566709518432617 Domain: biomedical Document type: Study Language: en Stimulation of human monocyte-derived macrophages (MDMs) with LPS or plasma samples from ARDS patients, categorized as either low-inflammatory or high-inflammatory phenotypes, in conjunction with treatment using mesenchymal stem cell-conditioned medium (CM) or extracellular vesicles (EVs), has led to the conclusion that MSCs regulate macrophages through the miR-181a-PTEN-pSTAT5-SOCS1 signaling axis ( 90 ). By altering the spatial distribution of MSCs and macrophages within a scaffold, the ‘cross-talk’ between these two cell types can be effectively manipulated, enabling controlled modulation of the scaffold-mediated bone immune response. In comparison to other multicellular models, the Taiji model, featuring a 2:1 ratio of MSCs to macrophages, demonstrated superior efficacy in activating anti-inflammatory M2 macrophages, enhancing osteogenic differentiation of MSCs, and accelerating new bone formation in vivo . This may be attributed to the activation of BMP-Smad, Oncostatin M (OSM), and Wnt/β-catenin signaling pathways in MSCs, mediated by macrophage-derived paracrine signaling factors ( 91 ). Wang et al. conducted indirect co-culture experiments with bone marrow-derived mesenchymal stem cells (BM-MSCs) inoculated on nanoporous titanium (Ti) scaffolds and macrophages. By blocking exosome secretion or extracting purified exosomes, they independently induced macrophage polarization. Their results indicated that, under the influence of TiO2 nanoporous topography, BM-MSCs can induce M1 polarization of macrophages, which may adversely affect the osteogenic microenvironment around implants ( 92 ). In vitro studies by Deng et al. further demonstrated that biotin-modified MSC-derived extracellular vesicles (Bio-EXs) are efficiently taken up by macrophages and exert immunomodulatory effects similar to those of unmodified MSC-EXs. Moreover, the Bio-GelMA@Bio-EX hydrogel sustained the release of MSC-EXs for seven days, promoting the polarization of macrophages towards the M2 phenotype ( 93 ). These findings collectively highlight the complex interplay between MSCs and macrophages in modulating the bone immune response and provide insights into the potential therapeutic applications of MSC-derived extracellular vesicles and hydrogels in bone regeneration and immune modulation. Section title: Immunomodulatory effects of MSC-EVs on DCs Educational score: 4.485488414764404 Domain: biomedical Document type: Study Language: en Dendritic cells (DCs) are antigen-presenting cells that fall within the category of innate immune cells. They possess the capability to detect antigenic substances from the external environment and present them on their cell surface to T cells. The immunoregulatory effects of MSCs on DCs are primarily characterized by inhibiting the differentiation, maturation, and antigen presentation of DCs, as well as reducing the expression of pro-inflammatory factors ( 94 ). In a model of T1DM, MSCs have been found to induce the secretion of IL-10 by immature DCs in vitro , thereby intercepting the initiation and expansion of autoreactive T cells in tissue inflammation ( 95 ). Furthermore, in streptozotocin (STZ)-induced diabetic mice, extracellular vesicles derived from mouse adipose-derived mesenchymal stem cells (mADSC-EVs) significantly diminished the inflammatory response involving DCs. These vesicles were also observed to continuously upregulate the protein expression of the NGF/TrkA pathway and decrease theLPS-mediated expression of IL-6 and TNF-α ( 96 ). These findings suggest that MSCs and their derived extracellular vesicles play a crucial role in modulating the immune response, particularly in the context of autoimmune diseases such as T1DM. By targeting DCs, MSCs and their derivatives may offer promising therapeutic strategies for the treatment of these conditions. Section title: Immunomodulatory effects of MSC-EVs on NKs Educational score: 4.434791088104248 Domain: biomedical Document type: Study Language: en Natural killer cells (NKs) constitute a vital component of the innate immune system, providing swift responses to virus-infected and tumorigenic cells ( 97 ). MSCs exhibit the capacity to modulate NK cell activity by inhibiting their proliferation, disrupting their cytotoxicity, and suppressing the secretion of proinflammatory cytokines. Investigations into the effects of soluble factors derived from adipose-derived mesenchymal stem cell (ADSC) media, collectively termed the ADSC secretome, on NK cells have revealed intriguing findings. Specifically, the presence of ADSC secretome was found to attenuate IL-2 signaling in IL-2-activated NK cells by inhibiting the phosphorylation of JAK1 and JAK3 kinases. Concurrently, the induction of CIS (cytokine-inducible Src homology 2 domain-containing protein) and DUSP4 (dual-specificity phosphatase 4) was enhanced ( 98 ). These results suggest that the ADSC secretome plays a pivotal role in modulating NK cell activity through the regulation of IL-2 signaling and the induction of CIS and DUSP4. This modulation may have important implications for the development of therapeutic strategies targeting NK cell-mediated immune responses in various diseases. Section title: Immunomodulatory role of MSCs in autoimmune inflammatory diseases Educational score: 4.0174031257629395 Domain: biomedical Document type: Review Language: en Traditional treatments for autoimmune inflammatory diseases commonly involve the administration of immunosuppressive drugs, corticosteroids, and nonsteroidal anti-inflammatory drugs. These medications are designed to quell the hyperactive immune response and mitigate inflammation. However, they frequently entail adverse side effects and may not yield satisfactory results in all patients. MSCs have emerged as a promising therapeutic alternative for autoimmune inflammatory diseases, thanks to their immunomodulatory capabilities. The integration of conventional therapies with MSCs presents a hopeful strategy for addressing autoimmune inflammatory diseases. Conventional treatments can swiftly manage inflammation and alleviate symptoms, whereas MSCs can offer sustained immunomodulation and facilitate tissue repair. By merging these two methodologies, it may be feasible to attain superior treatment outcomes and decrease the reliance on high doses of immunosuppressive drugs. We summarized the completed and ongoing clinical trials of MSCs in the treatment of autoinflammatory diseases in Table 1 . The results of these registered studies all demonstrate the safety and efficacy of MSCs in the treatment of autoimmune diseases. Section title: Immune regulation of T cells by MSCs in autoimmune diseases Educational score: 3.0923964977264404 Domain: biomedical Document type: Other Language: en Currently, the utilization of PD-L1 inhibitors in autoimmune diseases is still in its nascent research phase. A deeper understanding of the mechanism and suitable conditions for PD-L1 inhibitors in various autoimmune diseases is imperative, and further clinical trials are essential to validate their efficacy and safety. Section title: Immune regulation of T cells by MSCs in autoimmune diseases Educational score: 4.454226016998291 Domain: biomedical Document type: Study Language: en Several animal studies have delved into the potential of mesenchymal stem cells (MSCs) in treating various autoinflammatory diseases. For example, MSCs with high PD-L1 expression have demonstrated improvements in hyperglycemia and obesity in STZ-induced T1DM mice ( 85 ). Additionally, MSCs derived from decidual teeth have been found to restore the Treg and Th17 cell balance via the PD-L1/PD-1 pathway, leading to reduced glandular inflammation and alleviation of dry symptoms in mice with Sjögren’s syndrome ( 99 ). In models of graft-versus-host disease (GVHD), MSC treatment has been shown to lower clinical scores and extend survival in mice, with these benefits being reversed by PD-L1-specific antibodies both in vivo and in vitro ( 44 ). In a mouse model of classical experimental autoimmune uveitis (EAU), Yuan et al. discovered that overexpression of CCR5, a CCL5 receptor, in MSCs enhanced their homing ability and improved their immunomodulatory effects in preventing EAU by decreasing the number of infiltrating T cells and activated microglial cells, inhibiting the activation of the Nlrp3 inflammasome, and improving their immunomodulatory effect in preventing EAU ( 49 ). Wei et al. administered 1×10 6 hUC-MSCs derived from umbilical cord tissue intravenously to mice with autoimmune hepatitis (AIH) and assessed liver function and inflammation by measuring serum levels of alanine aminotransferase and aspartate aminotransferase, as well as pathological damage to liver tissue. They observed a significant decrease in CD4 + T-cell infiltration in the liver and a reduction in the frequency of IFNγ- and IL-17A-producing CD4 + T-cells in the spleen, along with a trend towards an increase in Treg cells in liver tissue. RNA sequencing analysis of liver tissues revealed significant negative regulation of inflammation-related signaling pathways in the UC-MSC-treated group, suggesting that hUC-MSCs have the potential to suppress the immune response and provide a potential clinical option for ameliorating AIH ( 100 ). Wu et al. found that infusion of TGFBI-knockout hUC-MSCs had an impaired therapeutic effect on T1DM mice, increasing T-cell infiltration and the expression of IFN-γ and IL-17A in the spleen. They also discovered that TGFBI derived from hUC-MSCs inhibited the proliferation of activated T cells by interfering with the expression of the G1/S checkpoint CyclinD2, suggesting that TGFBI may be a new target for T1DM therapy ( 101 ). Section title: Immunoregulation of MSCs to other immune cells in autoimmune diseases Educational score: 4.158093452453613 Domain: biomedical Document type: Study Language: en Furthermore, the application of human adipose-derived MSCs has the potential to diminish the infiltration and accumulation of T cells and CXCL9 + macrophages within the islet beta cell area ( 46 ). Moreover, MSCs engineered to express CEACAM1 have demonstrated efficacy in ameliorating inflammatory manifestations and enhancing survival rates in GVHD mouse models, achieved by mitigating NK cell activation and cytotoxicity, as well as suppressing T cell proliferative activation ( 60 ). Section title: Immunomodulatory function by paracrine activity Educational score: 4.530510425567627 Domain: biomedical Document type: Study Language: en In preclinical studies involving mouse models of dextran sulfate sodium (DSC)-induced ulcerative colitis (UC) and imiquimod (IMQ)-induced psoriasis, the researchers demonstrated that mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) expressing PD-L1 effectively inhibited inflammatory immune cells, targeting and repairing tissue damage through the PD-1/PD-L1 pathway. The results of this assay revealed that MSC-sEVs-PD-L1 suppressed the proliferation of T cells, dendritic cells, and macrophages, leading to a reconfiguration of the local immune microenvironment via the induction of regulatory T cells and the elimination of effector T cells, accompanied by modulation of cytokine expression ( 102 ). Given its straightforward preparation, low cost, feasibility, and biosafety, the researchers believe this technique holds promising potential for clinical application. In the realm of GVHD, extracellular vesicles derived from mesenchymal stem cells (MEXs) have been shown to alleviate clinical symptoms and extend the survival of recipient mice by promoting the differentiation of Treg cells in the spleen while preserving the cytotoxic anti-leukemia effect of recipient mouse CD8 + T cells ( 94 ). Compared to the mechanism of intercellular contact, the paracrine mechanism of MSCs has been extensively studied across various clinical, animal, and cellular research studies, particularly focusing on the regulation of T cells and macrophages. Based on multicellular organ tissues established in Mdr2 -/- mice and patients with primary sclerosing cholangitis (PSC), Chen et al. proposed a potential therapeutic role for exosomes derived from human placenta-derived mesenchymal stem cells (ExoMSC) in hepatic fibrosis associated with PSC or Th17-related diseases. Animal studies showed that ExoMSC ameliorated hepatic fibrosis in Mdr2 -/- mice, significantly reducing collagen deposition in the precatheteric region, inhibiting Th17 differentiation, and decreasing the proportion of CD4 + IL-17A + T cells in vivo . Multicellular organ tissue studies further revealed that ExoMSC alleviated the hypersecretory phenotype and cell-cell interactions in the hepatic Th17 microenvironment by modulating PERK/CHOP signaling ( 76 ). Section title: Immune regulation of T cells by MSCs in autoimmune diseases Educational score: 4.386561393737793 Domain: biomedical Document type: Study Language: en A study conducted in vivo revealed that a dexamethasone-based liposomal integrated MSCs (Dexlip-MSCs) treatment approach significantly extends the drug’s circulation time in the body and reduces the necessary dosage compared to dexamethasone alone. Dexlip-MSCs were found to upregulate the expression of CRISPLD2 and other anti-inflammatory factors by activating the glucocorticoid receptor signaling pathway. This led to a decrease in pro-inflammatory factors and an enhanced anti-inflammatory inhibitory effect on CD4 + T cells, ultimately mitigating the progression of systemic lupus erythematosus (SLE) in MRL/lpr mice ( 25 ). In a separate clinical trial involving 30 patients with conventionally refractory active SLE, umbilical cord-derived mesenchymal stem cells (UCMSCs) transfusions were administered. The results showed an upregulation of Treg cell expression and a downregulation of Th17 expression through TGF-β and PGE2, effectively alleviating the clinical symptoms of SLE patients. These findings suggest a promising new approach for MSCs as adjuvant therapy for SLE ( 103 ). However, in the NCT01539902 clinical trial indicates that hUC-MSC has no significant additional effects beyond standard immunosuppression. Eighteen patients with WHO classification III or IV lupus nephritis (LN) were randomly assigned to hUC-MSCs (2×10 8 cells) or placebo. All patients received standard immunosuppressive therapy, including intravenous methylprednisolone and cyclophosphamide, followed by oral prednisolone and mortemycophenolate. Nine of 12 patients (75%) in the hUC-MSC group experienced a response, compared with five of six patients (83%) in the placebo group. The proportion of patients achieving a complete response was similar in both groups, along with similar improvements in serum albumin, complement, kidney function, systemic lupus erythematosus disease activity index and the British Isles Lupus Assessment Group score. The trial was abandoned after enrolling 18 patients when it became clear it would not show a positive therapeutic effect ( 104 ). Li et al. conducted trial NCT01741857 and found that circulating miR-320b and MAP3K1 may be involved in the proliferation of SLE CD4 + T cells. Small RNA sequencing analysis revealed a significant decrease in circulating miR-320b levels in SLE patients after mesenchymal stem cell transplantation (MSCT). In vitro experiments further showed that reduced levels of MAP3K1 in SLE peripheral blood mononuclear cells (PBMCs) were associated with CD4 + T cell proliferation. In MRL/Lpr mice, miR-320b overexpression exacerbated SLE symptoms, while inhibition of miR-320b promoted disease remission ( 105 ). Section title: Immune regulation of T cells by MSCs in autoimmune diseases Educational score: 4.433422088623047 Domain: biomedical Document type: Study Language: en In the NCT01941394 clinical trial, patients undergoing allogeneic hematopoietic stem cell transplantation were randomized into two groups: one receiving standard GVHD prophylaxis and the other also receiving MSC infusion when leukocytes recovered to 1,000 cells/μL (transplantation, day E0). After 30 days, patients injected with MSCs showed significantly increased CD4 + T-cell counts, as well as increased levels of IL-6, IL-8, IL-17, TNF-α, and IFN-γ. The concentrations of G-CSF, GM-CSF, PDGFbb, FGFb, and IL-5 also increased on day E+30. Regardless of MSC administration, there was a significant increase in the number of CD8 + cells at day E+30, while the concentrations of IL-9, eotaxin, IP-10, MCP-1, and MIP-1a were all increased at day 30. These results suggest a positive effect of MSC administration on the recovery of T-cell subsets and the immune system in patients after allogeneic HSCT ( 106 ). García-Berna et al. conducted a controlled HCELL-MSC/HCELL + MSCs administration assay using a mouse model of acute graft-versus-host disease (aGVHD). The results suggested that MSC surface HCELL/CD44 connections significantly enhanced the immunomodulatory activity of MSCs by inducing the secretion of multiple potent immunomodulatory molecules, including IL-10 ( 107 ).The efficacy and safety of umbilical cord mesenchymal stem cells was demonstrated in a phase 2 study to prevent chronic graft-versus-host disease after haploid hematopoietic stem cell transplantation. The results suggest that repeated MSCs infusion may suppress aGVHD symptoms in HLA-haplo HSCT patients, accompanied by changes in T, B, and NK cell numbers and subtypes, resulting in immune tolerance ( 108 ). In psoriasis, co-culture of DPMSCs with CD3 + T cells demonstrated inhibitory effects on T cell proliferation and induction of apoptosis. DMSCs also decreased the Th17/Treg cell ratio and enhanced Treg-mediated inhibition of effector T cells. These effects were partially attenuated by TGF-β receptor inhibitors, indicating a potential therapeutic strategy for ameliorating psoriasis symptoms ( 48 ). Furthermore, sFgl2 gene-modified MSCs were found to promote Treg cell differentiation, inhibit Th17 and Th1 cell differentiation, and exhibit a strong therapeutic effect in an experimental model of autoimmune hepatitis induced by con A ( 50 ). UCMSC-derived exosomes were shown to reduce elevated levels of autophagy in peripheral blood CD4 + T cells of patients with primary Sjögren’s syndrome. These exosomes regulated T cell proliferation and apoptosis, inhibited the differentiation of Th17 cells, and promoted the differentiation of Treg cells, ultimately restoring the Th17/Treg cell balance in these patients ( 74 ). In vitro studies have shown that TLR3 stimulation can specifically enhance the immunosuppressive effects of MSCs on the proliferation and differentiation of Th1 and Th17 subtypes, while TLR4 agonists can completely reverse these effects ( 109 , 110 ). Kaur et al. compared MSC-EV generated from monolayer cultures (ML-EV) or microcarrier cultures (MC-EV) using a mouse model of experimental autoimmune uveitis (EAU) and other in vitro assays reflecting the in vivo mode of action of MSC-EV. The results showed that MC-EV, carrying a high concentration of TGF-β1 EV, had greater efficacy in blocking disease progression, inducing apoptosis, and inhibiting retinal-reactive T cell chemotaxis in EAU mice compared to ML-EV. They also proposed a mechanism by which MSC-EV, as well as treatment with TGF-β1 or let-7b, could inhibit the MAPK/ERK pathway of activated T cells ( 77 ). Wang et al. found that human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSCs-EVs) could alleviate dry eye disease (DED) symptoms by multi-targeting the IRAK1/TAB2/NF-κB pathway through certain miRNAs. In the DED model induced by a dry environment combined with scopolamine administration, treatment with hUCMSC-EVs increased tear volume and maintained corneal integrity in DED mice. It also decreased the levels of proinflammatory cytokines in tears, increased the density of microglandular vesicle cells, and inhibited apoptosis and CD4 + cell infiltration ( 111 ). Chen et al. compared intestinal Peyer’s patches (PP)-derived MSCs with bone marrow-derived MSCs and systematically demonstrated the prominent benefits of mouse MSC proteins in the treatment of inflammatory bowel disease (IBD) in mice, partly through IL-22 ( 112 ). Related clinical studies have demonstrated the safety of MSCs therapy. Specifically, a phase I clinical trial involving Cymerus mesenchymal stem cells (CYP-001) was conducted at seven centers in the UK and Australia to address steroid-resistant aGVHD (SR-aGVHD) in adults post-allogeneic hematopoietic stem cell transplantation. Participants were stratified into two groups: Group A (n=8) received two intravenous infusions of CYP-001, one on day 0 and one on day 7, at a dose of 1×10 6 cells per kilogram of body weight, capping at a maximum absolute dose of 1×10 8 cells. Group B (n=7) similarly received infusions on days 0 and 7, but at an escalated dose of 2×10 6 cells per kilogram, with a maximum absolute dose of 2×10 8 cells. Notably, no participants received any other investigational drugs within 28 days of prior to the first CYP-001 dose. Reporting the 2-year follow-up results, we found that 9 out of 15 participants (60%) survived, a figure more favorable than those reported in previous SR-aGVHD studies. The causes of death were complications commonly observed in allogeneic hematopoietic stem cell transplant recipients and were deemed unrelated to CYP-001 treatment by the investigators. Furthermore, no serious adverse events, tumors, or other safety concerns associated with CYP-001 were reported ( 113 ). Section title: Immunoregulation of MSCs to other immune cells in autoimmune diseases Educational score: 4.436578750610352 Domain: biomedical Document type: Study Language: en Ranjbar and colleagues conducted a Phase I trial involving nine patients with biopsy-confirmed, standard therapy-refractory LN. They administered a systemic infusion of 2×10 6 allogeneic adipose-derived MSCs (AD-MSCs) per kilogram of body weight and monitored the patients for 12 months. The follow-up results indicated that allogeneic AD-MSC transplantation exhibited a favorable safety profile and was effective in decreasing urinary protein excretion and reducing disease activity. Notably, the most significant improvements in proteinuria and disease activity scores were observed at 1 and 6 months post-intervention ( 114 ). Furthermore, research has delved into the immunomodulatory mechanisms of MSCs, particularly their ability to regulate macrophages. The administration of human umbilical cord-derived MSC-derived extracellular vesicles (hUC-MSCs-EVs) induced macrophages to adopt an anti-inflammatory M2 phenotype. These induced M2 macrophages demonstrated a robust capacity to inhibit CD4 + T cell proliferation and promote the generation of Tregs in vitro , thereby reducing inflammation and mitigating tissue damage. Additionally, MSCs pretreated with the TLR5 agonist KMRC011 exhibited enhanced immunosuppressive effects on lymphocyte proliferation in both in vivo and in vitro mouse GVHD models. This pretreatment increased the secretion of immunosuppressive cytokines such as IDO and COX2, and enhanced the expression of M2 macrophage polarization cytokines, including M-CSF and IL-10 in vitro . This treatment facilitated the polarization of macrophages towards the M2 phenotype, thereby increasing the proportion of anti-inflammatory cells and alleviating GVHD severity in mice. These findings present a novel perspective for GVHD treatment ( 84 ). Moreover, studies have emphasized the significance of MHCI on MSCs in NK cell-mediated killing and immunosuppression ( 115 ). MEXs have been shown to inhibit the expression of costimulatory molecules and the secretion of cytokines derived from DCs ( 94 ). Exos@SFMA have proven effective in alleviating synovial inflammation and joint destruction by markedly reducing the responsiveness of follicular helper T cells and further inhibiting the differentiation of germinal center B cells into plasma cells ( 39 ). A randomized Phase 2 trial conducted at 15 sites in nine countries tested the safety and activity of MSC in the treatment of multiple sclerosis. 144 patients with active relapsing-remitting or progressive multiple sclerosis were randomly assigned according to a cross-over design, with one group (n=69) receiving a single intravenous injection of autologous bone marrow-derived MSCs followed by placebo at week 24 and the other group (n=75) receiving placebo at week 24 followed by autologous MSCs. They were followed up at 48 weeks. 213 adverse events were recorded, again distributed between groups (93 were recorded in 35 of 69 patients who received MSC for the first time, compared with 120 in 42 of 75 patients who received placebo for the first time). The most commonly reported adverse events were infection and infestations, with 54 of 213 adverse events (18 of 93 in the early MSCs group and 36 of 120 in the late MSCs group). Nine serious adverse events were reported among seven patients in the placebo group, while none were reported in the MSC group. All serious adverse events were considered unrelated to the therapeutic infusion. No deaths were reported during the study period. However, no effect was shown on GEL (an MRI surrogate marker for acute inflammation) in patients with active MS at week 24, so the conclusions of this study do not support the use of bone marrow-derived MSCS in the treatment of active MS ( 116 ). Section title: Discussion Educational score: 3.5034236907958984 Domain: biomedical Document type: Other Language: en MSCs exhibit the potential to significantly enhance the effectiveness of conventional therapies by mitigating drug resistance and alleviating adverse side effects. Their ability to promote the regeneration of damaged tissues further underscores their importance, as it can lead to improved functional recovery and enhanced quality of life for patients. Section title: Immunomodulatory characteristics of MSCs Educational score: 4.156472682952881 Domain: biomedical Document type: Review Language: en MSCs exert their immunomodulatory effects through direct cell contact with immune cells, such as T cells, B cells, macrophages, and dendritic cells, as well as through the secretion of paracrine mediators. This dual mode of action allows MSCs to regulate the activation, proliferation, and differentiation of immune cells, thereby modulating the immune response. The production of cytokines, growth factors, and chemokines by MSCs further enhances their immunomodulatory capacity, promoting tissue repair and regulating the inflammatory environment. Section title: Immunomodulatory characteristics of MSCs Educational score: 4.298699378967285 Domain: biomedical Document type: Review Language: en MSCs significantly impact T cell activity by suppressing activation and proliferation through the PD-1/PD-L1 pathway, an immune checkpoint mechanism. Pretreatment with inflammatory cytokines enhances MSCs’ immunosuppressive effects, suggesting dynamic immunomodulation. MSCs also regulate B cell function by inhibiting proliferation and antibody production, affecting IL-10 production and B cell cycle progression. They interact with innate immune cells, reprogramming macrophages and influencing NK cell activation and cytotoxicity. MSCs affect DCs maturation and function, impacting both innate and adaptive immune responses. These findings highlight MSCs’ potential as an immunotherapeutic tool, but further research is needed to fully understand the mechanisms and explore clinical applications. Section title: Immunomodulatory characteristics of MSCs Educational score: 4.3174028396606445 Domain: biomedical Document type: Review Language: en The paracrine activity of MSCs has emerged as a pivotal mechanism through which these cells exert their immunomodulatory effects on various immune cell populations. By secreting cytokines and exosomes, MSCs can influence both innate and adaptive immune responses, making them a promising therapeutic tool in modulating immune dysfunction. The studies reviewed provide compelling evidence that MSC-EVs regulate T cell proliferation and differentiation, suppressing Tc and Th1 cells while enhancing Tregs and anti-inflammatory IL-10 ( 71 , 75 , 89 , 117 ). MSC-EVs also regulate T cell signaling pathways, such as inhibiting the MAPK/ERK pathway and modulating PERK/CHOP signaling ( 77 ). This shift towards a tolerogenic immune environment suggests a therapeutic role in autoimmune and inflammatory disorders. MSCs and their EVs have multifaceted immunomodulatory effects on macrophages, DCs, and NK cells, offering therapeutic potential in autoimmune diseases and cancer immunotherapy. Future research should focus on elucidating mechanisms and exploring therapeutic applications. Understanding the mechanisms of MSCs-mediated immune modulation will facilitate the development of targeted therapeutic strategies. MSCs and their EVs, especially PD-L1-expressing sEVs, show promise in preclinical and clinical studies for treating inflammatory and autoimmune diseases. Section title: Exploration of application in animal model Educational score: 4.148827075958252 Domain: biomedical Document type: Study Language: en In mouse models of UC and psoriasis, MSC-sEVs-PD-L1 suppressed immune cell proliferation, induced Tregs, and modulated cytokines, facilitating tissue repair. MSCs’ paracrine mechanism, studied extensively, also helps regulate T cells and macrophages, with MEXs alleviating GVHD symptoms in mice ( 94 ). Exosomes from placenta-derived MSCs ameliorate hepatic fibrosis in animal studies. In SLE, MSCs and their derivatives show variable results as adjuvant therapy, possibly due to patient differences. MicroRNA modulation, like miR-320b in SLE CD4 + T cells, suggests novel therapeutic strategies ( 105 ). MSCs also aid immune reconstitution after HSCT and enhance immunomodulatory activity in GVHD mouse models. Further clinical trials are needed to validate these therapies. Section title: Clinical trials and emerging trends Educational score: 4.370634078979492 Domain: biomedical Document type: Review Language: en A multitude of clinical trials have been initiated to rigorously assess the safety and efficacy of MSCs in the management of autoimmune inflammatory diseases. These trials have yielded encouraging results, demonstrating that MSCs can ameliorate symptoms, diminish disease activity, and elevate patients’ overall quality of life. The studies discussed highlight the therapeutic potential and safety of MSCs and their derivatives in treating immune-mediated diseases. DPMSCs show efficacy in preventing GVHD, psoriasis, and autoimmune diseases by modulating immune cell populations and T cell differentiation ( 89 , 118 , 119 ). Gene-modified MSCs and exosomes also exhibit therapeutic effects. Research explores enhancing MSCs’ immunosuppressive effects through TLR stimulation and different culture methods for generating MSC-EVs. MC-EVs show promise in blocking disease progression and alleviating symptoms A phase I trial supports the safety of MSCs in treating GVHD. Ranjbar’s study demonstrates the safety and efficacy of allogeneic AD-MSCs in reducing LN symptoms, with sustained therapeutic effects ( 114 ). Huang et al. conducted an open-label, multicenter, parallel randomized clinical trial to evaluate the efficacy of repeated umbilical cord MSCs infusions during the early post-transplant period (days 45 and 100) for preventing chronic graft-versus-host disease (cGVHD) after haploidentical hematopoietic stem cell transplantation (haplo-HSCT). The results of this trial reveal that early repeated MSCs infusions decrease the incidence and severity of cGVHD and enhance graft-versus-host disease-free and relapse-free survival (GRFS) among patients, without raising the incidence of adverse events ( 120 ). A total of 203 patients with steroid-resistant acute graft-versus-host disease (SR aGVHD) from nine centers in China took part in a multicenter, randomized, open-label phase 3 clinical trial to evaluate the efficacy and safety of MSCs combined with basiliximab and calcineurin inhibitors as second-line therapy for SR aGVHD. The trial results demonstrate that the combination of MSCs with second-line treatment improves therapeutic outcomes, reduces drug toxicity and the incidence of cGVHD, without increasing the risk of relapse, and is well-tolerated, highlighting the promising therapeutic potential of MSC-based combination therapy ( 121 ). The immunomodulatory mechanisms MSCs, especially their capacity to regulate macrophages, are of interest. Pretreating of MSCs with a TLR5 agonist intensifies their immunosuppressive effects, representing a promising strategy for GVHD treatment ( 84 ). The significance of MHC I on MSCs in NK cell-mediated killing and immunosuppression is also noted ( 115 ). Exos@SFMA alleviates synovial inflammation in arthritis models ( 39 ). However, a phase 2 trial in multiple sclerosis showed mixed results, indicating that further research is required ( 116 ). Section title: Clinical trials and emerging trends Educational score: 3.852874517440796 Domain: biomedical Document type: Review Language: en Despite these promising findings, there remains a pressing need for additional research to refine the integration of MSCs with conventional therapies. Future endeavors should concentrate on pinpointing the most suitable cell source, determining the optimal dosage, and establishing the most effective route of administration for MSCs. Equally important is the continued evaluation of the long-term safety and sustained efficacy of this innovative therapeutic approach. Section title: Clinical trials and emerging trends Educational score: 4.018978595733643 Domain: biomedical Document type: Review Language: en In summation, the synergistic combination of conventional therapies and MSCs stands as an auspicious strategy in the treatment of autoimmune inflammatory diseases. This paradigm shift holds the potential to elevate treatment outcomes and alleviate the considerable burden these diseases impose on patients. As we advance, it is imperative to delve deeper into the intricate mechanisms of action of MSCs and to optimize their utilization within the clinical setting, ultimately paving the way for more targeted and effective therapeutic interventions. Overall, MSCs offer promising therapeutic options for immune-mediated diseases, with immune modulation and tolerance induction as potential mechanisms. Further research and larger trials are required to confirm efficacy and safety in different patient populations and disease contexts. | Review | biomedical | en | 0.999998 |
PMC11695408 | Section title: Introduction Educational score: 3.4037563800811768 Domain: biomedical Document type: Other Language: en The global pandemic of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS−CoV−2) (one newly defined respirovirus), has impacted populations globally since its emergence in late 2019. With the advent of new viral variants, repeat infections are becoming increasingly prevalent, and there is growing evidence to suggest that these may heighten the risk of developing long COVID . Section title: Introduction Educational score: 4.15041971206665 Domain: biomedical Document type: Review Language: en Pregnant women are typically more susceptible to infections due to physiological changes during pregnancy, and they represent a particularly vulnerable group during such infectious disease outbreaks. In fact, COVID-19 has been shown to result in more severe morbidity in pregnant women as compared to non-pregnant populations . Increasing evidence has demonstrated adverse outcomes associated with SARS-CoV-2 infection during pregnancy, which includes preterm birth, stillbirth, and maternal mortality . In addition to understanding the implications of respirovirus with vertical transmission, such as Zika virus, which has led to significant fetal health complications, it is important to consider SARS-CoV-2, a novel respiratory virus that also raises concerns regarding potential vertical transmission . The evidence for SARS-CoV-2 vertical transmission is still evolving, but studies have indicated possible transmission from mother to fetus . Consequently, the identification of risk factors for repeat infections in pregnant women has emerged with the highest public health priority for mitigating both immediate impacts and long-term sequelae such as long COVID. Section title: Introduction Educational score: 4.105580806732178 Domain: biomedical Document type: Study Language: en One potential risk factor for COVID-19 is exposure to air pollution . As a dominant component of air pollutants, the ground-level ozone is usually formed by chemical reactions between nitrogen oxides, volatile organic compounds, methane, and carbon monoxide in the presence of sunlight . However, ozone exposure during pregnancy has been shown to cause oxidative stress, inflammation, and endothelial dysfunction . At the molecular level, ozone can impair placental growth factor signaling and increase anti-angiogenic factors, which is causally associated with dysregulations of placental vascular development . Numerous studies have linked gestational ozone exposure with adverse birth outcomes, including low birth weight, small for gestational age, and preterm birth . However, the mechanisms linking ozone exposure and adverse pregnancy outcomes are not fully understood. Section title: Introduction Educational score: 4.110787868499756 Domain: biomedical Document type: Study Language: en Although previous studies have examined the impacts of ozone exposure in pregnancy, few examined the relationship between longitudinal ozone exposure during pregnancy and SARS-CoV-2 infection in late pregnancy, and this represents a significant gap in the literature that requires immediate attention. It is plausible that ozone-induced placental dysfunction alters the susceptibility to viral infections, but more research is critically needed to investigate this hypothesis further. Therefore, in this retrospective cohort study, we aimed to assess the association between longitudinal ozone exposure over gestation and the risk of testing positive for SARS-CoV-2 in late pregnancy, which will help elucidate whether air pollution is a novel risk factor for COVID-19 in pregnant women and provide insights into the mechanisms linking ozone and SARS-CoV-2 infection. Section title: Population Educational score: 4.039311408996582 Domain: biomedical Document type: Study Language: en This study was conducted in Jinan, east China between November 3, 2022 and January 6, 2023, encompassing one month before and after the Chinese government easing of COVID-19 control measures. The study population consisted of 600 pregnant women with singleton births who were recruited during this period, of whom 300 were infected with SARS-CoV-2 and 300 were not. Detailed maternal information was extracted from electronic medical records and questionnaires, including age, pre-pregnancy body mass index (BMI), education background, vaccination status, gestational diabetes mellitus (GDM), folic acid supplementation, medication use during pregnancy, age at first pregnancy, gravidity, prior preterm births or abortions, age at menarche, menstrual cycle characteristics (days of menstruation, cycle length, regularity), gestational age at delivery, conception method, SARS-CoV-2 infection status, and infant sex. Exclusion criteria were set as follows: residence outside the study area, neonate congenital disabilities, and maternal age at delivery outside 18-50 years. All participants denied any history of alcohol, tobacco, or drug use. The study protocol was approved by the Ethics Committee of the Maternal and Child Health Care Hospital of Shandong Province Affiliated to Qingdao University in Jinan, China . Section title: Exposure Educational score: 4.050518035888672 Domain: biomedical Document type: Study Language: en In accordance with our previous study , we monitored ground-level ozone in the designated study area on an hourly basis using publicly accessible data at the Air Quality and Pollution Measurement website ( https://aqicn.org/ ), which is part of the World Air Quality Index project that commenced in 2007. We calculated the gestational atmospheric ozone exposure levels by averaging daily ozone concentrations from the onset of pregnancy until delivery. Simultaneously, we gathered data on local temperature, wind direction and wind speed from the NOAA Integrated Surface Database (ISD) via the worldmet package in R software. To examine the long-term trend of atmospheric ozone from 2015 to 2023, we conducted Theil-Sen trend statistical analysis, where the openair package was employed to analyze and illustrate variations in atmospheric ozone on an hourly and daily basis. Section title: Outcomes and covariates Educational score: 4.11594820022583 Domain: biomedical Document type: Study Language: en The primary outcome of this study was SARS-CoV-2 infection rate, which was detected through reverse transcription polymerase chain reaction (RT-PCR) testing of nasal and pharyngeal swabs according to the instructions provided by the commercial PCR kit. The assay targeted the ORF1ab, N, and E genes of the SARS-CoV-2 virus. Specific primer sequences used for amplification were as follows. RdRP, forward primer: GTGARATGGTCATGTGTGGCGG, reverse primer: CARATGTTAAASACACTATTAGCATA; N, forward primer: CACATTGGCACCCGCAATC, reverse primer: GAGGAACGAGAAGAGGCTTG; E, forward primer: ACAGGTACGTTAATAGTTAATAGCGT, reverse primer: ATATTGCAGCAGTACGCACACA. Section title: Outcomes and covariates Educational score: 4.0167365074157715 Domain: biomedical Document type: Study Language: en For study subjects, we randomly recruited them at 29-40 gestational weeks. Covariates were chosen based on previous studies and clinical experience, including gestational age, pre-pregnancy BMI, education background, vaccination status, GDM, folic acid supplementation, medication use during pregnancy, age at first pregnancy, gravidity, prior preterm births or abortions, age at menarche, menstrual cycle days and length, gestational age at delivery, conception method, and infant sex. Of them, gestational age, pre-pregnancy BMI, age at first pregnancy, age at menarche, menstrual cycle length, and gestational age at delivery were continuous variables, while the education background, vaccination status, GDM, folic acid supplementation, medication use during pregnancy, menstrual cycle regularity, conception method, and infant sex were categorical variables. Noteworthy, we collected hourly atmospheric ozone measurements, calculated daily averages, then gestational averages, and included them as continuous variables in our analyses. Section title: Statistical analysis Educational score: 3.4982550144195557 Domain: biomedical Document type: Study Language: en Continuous demographic variables and atmospheric ozone exposure data were presented as mean and standard deviation. Categorical variables were showed as frequency with corresponding percentage. All study subjects were divided into two groups based on the infection status, with 300 subjects each group. Differences between groups were described and compared using t-test, chi-square test, or Fisher’s exact test, as appropriate. Section title: Statistical analysis Educational score: 4.0736775398254395 Domain: biomedical Document type: Study Language: en To determine the effect of gestational ozone exposure on SARS-CoV-2 infection during late pregnancy, we employed both univariate and multivariate logistic regression analyses. Initially, we explored the univariate relationship between each variable and SARS-CoV-2 infection utilizing logistic regression analysis. Subsequently, we adjusted for potential confounding effects introduced by demographic variables using a multivariate logistic regression model. Simultaneously, we restructured the ozone exposure data into increments of interquartile ranges and incorporated it as a categorical increment in an alternate multivariate model. Prior to multivariate modeling, we scrutinized the collinearity between variables using correlation analysis . All analyses were executed in R 4.3.1. A p-value of less than 0.05 was deemed statistically significant unless stated otherwise. Section title: Variation of atmospheric ozone of the study area Educational score: 3.9998297691345215 Domain: biomedical Document type: Study Language: en To characterize the atmospheric ozone of the study area, we studied its longitudinal change from 2015 to 2023. As shown in Figure 1 , the Theil-Sen regression analysis revealed that the atmospheric ozone continuously grew at 1.85 ppb/year, ranging from 32.67 to 95.54 ppb over the study period. Besides, the annual atmospheric ozone peaked between April and October, at which time the wind blew from the south or southwest. Similarly, the daily ozone concentration changed in line with temperature, which was detected with the highest concentration at 14:00 in the noon and the lowest at 7:00 in the morning. Based on ozone data collected from each pregnancy period, we showed its dynamic variation through polar plots, where we defined the highest ozone concentration at wind directions of south and west, with relatively high local temperature in summer. Consistently, the cluster analysis identified two clusters of the high and low ozone clusters, and compared to the year 2021, the atmospheric ozone increased in 2022 with wind blowing from the south, southwest, and northeast, suggesting that the ozone of Jinan is worsening both in summer and winter. These data suggest that the atmospheric ozone in Jinan is worsening and changes tightly and periodically with local temperature. Section title: Baseline characteristics of study subjects Educational score: 4.083469867706299 Domain: biomedical Document type: Study Language: en A total of 600 pregnant women with singleton birth were included in this study ( Table 1 ), the age of whom varied between 19 and 47 years, with an average of 30.91 ± 4.26 years. Pre-pregnancy BMI values were also recorded, averaging at 26.81 ± 4.00 and spanning from 13.72 to 41.77. The women’s first pregnancies typically occurred around the age of 27.41 ± 3.63 years, with individual cases ranging from 16 to 42 years. Menarche was reported to have started at an average age of 13.79 ± 1.26 years, with a range of 11 to 32 years across the group. Menstrual cycles generally lasted for about 5.74 ± 0.91 days, with individual cycles ranging from 2 to 7.5 days in length. The gestational age at delivery averaged at 39.10 ± 2.07 weeks, with individual cases ranging from 32.00 to 41.43 weeks. Lastly, gestational ozone exposure levels were measured, averaging at 88.64 ± 3.34 ppb and ranging from 32.67 to 95.54 ppb across the group. Section title: Baseline characteristics of study subjects Educational score: 3.2856404781341553 Domain: biomedical Document type: Study Language: en In terms of categorical variables, a total of 580 subjects (80%) had a high school education or higher, 126 subjects (79%) received at least one vaccination, and 173 pregnant women were diagnosed with GDM, while 566 subjects received folic acid supplementation during their pregnancy. A total of 51 subjects took medication for conditions other than COVID-19 during their pregnancy, and while most subjects (572) conceived naturally, a minority (48) used artificial conception methods. Regarding the newborns, there were slightly more males (305) than females (295). Section title: Baseline characteristics of study subjects Educational score: 3.8648412227630615 Domain: biomedical Document type: Study Language: en Based on whether they were infected with SARS-CoV-2, all study subjects were divided into two groups, with each group containing 300 subjects. Compared to the uninfected pregnant women, those infected with SARS-CoV-2 had a higher pre-pregnancy BMI, vaccination rate, prevalence of GDM, and menstrual days, but a lower age at menarche and gestational ozone exposure level. There were no statistically significant differences between groups in terms of the remaining demographic variables (p > 0.05). Section title: Risk assessment of gestational ozone exposure on late pregnancy SARS-CoV-2 infection Educational score: 4.089241027832031 Domain: biomedical Document type: Study Language: en To investigate the association of gestational ozone exposure and late pregnancy SARS-CoV-2 infection, we conducted multivariate logistic regression analyses. The model considered the infection status as the dependent variable (infected vs. uninfected), and demographic characteristics and gestational ozone exposure data as independent variables. As depicted in Figure 2 , after adjusting for confounding effects of demographic variables, we found that each unit increase in gestational ozone exposure decreased 40% risk of late pregnancy SARS-CoV-2 infection [OR95%CI: 0.60(0.57, 0.66)]. Conversely, an increase in age at pregnancy and pre-pregnancy BMI was associated with an elevated risk of infection [OR95%CI: 1.06(1.00, 1.12) and 1.07(1.01, 1.13)]. Section title: Risk assessment of gestational ozone exposure on late pregnancy SARS-CoV-2 infection Educational score: 4.105905532836914 Domain: biomedical Document type: Study Language: en Moreover, we categorized the ozone data into four quartiles based on previous studies and incorporated them into the multivariate logistic regression model . Compared to the first quartile (32.7 to 87.1 ppb), the third (88.8 to 90.6 ppb) and fourth (90.6 to 95.5 ppb) quartiles were associated with a significantly lower risk of infection [OR95%CI: 0.20(0.12, 0.34) and 0.01(0.00, 0.03)]. Additionally, an increase in age at first pregnancy was linked decreased risk of infection [OR95%CI: 0.92(0.86, 0.99)], while an increase in age at pregnancy and pre-pregnancy BMI corresponded to a higher risk [OR95%CI: 1.07(1.01, 1.14) and 1.07(1.00, 1.13)]. The robustness of these two models was confirmed through receiver operating characteristic (ROC) analysis, yielding area under the curve (AUC) values of 0.842(0.809, 0.876) and 0.860(0.830, 0.890) . Section title: Discussion Educational score: 4.116435527801514 Domain: biomedical Document type: Study Language: en To study the effect of longitudinal gestational ozone exposure on respirovirus infection in late pregnancy, we conducted this retrospective cohort study, which involved 600 pregnant women who gave birth to singletons in Jinan, China, between January 2022 and March 2023. The atmospheric ozone levels in this study area were meticulously measured, along with individual ozone exposure levels during each trimester. Comprehensive data collection was undertaken, encompassing demographic, clinical, and laboratory data of the expectant mothers and their newborns. The findings revealed a concerning deterioration in Jinan’s atmospheric ozone, exhibiting fluctuations in sync with local temperature and wind directions. Interestingly, an inverse association was discovered between gestational ozone exposure and late pregnancy SARS-CoV-2 infection after adjusting for potential confounders, and this intriguing finding hints at a possible protective role of ozone against SARS-CoV-2 infection in pregnant women. Section title: Discussion Educational score: 4.261807441711426 Domain: biomedical Document type: Study Language: en While the precise mechanism of ozone’s antiviral activity remains to be fully elucidated, it is postulated that ozone may interact with viruses via a direct molecular ozone reaction pathway and/or indirectly through the generation of reactive oxygen species (ROS) such as hydroxyl radicals (·OH), superoxide anions (O2·-), and hydrogen peroxide (H2O2) as byproducts of ozone decomposition . In line with previous studies, the application of ozone to inactive SARS-CoV-2 had been well documented . For example, Angeles et al. reported that ozone can oxidize the viral envelope and RNA, rendering the virus unable to infect host cells . Particularly, previous studies have demonstrated the inactivation of a human coronavirus (HCoV-229E) and other related viruses by ozone under various conditions . However, ozone is also a hazardous gas that can cause lung damage if inhaled at high levels, and therefore strict safety protocols must be followed when using ozone as a disinfectant. Section title: Discussion Educational score: 4.370452404022217 Domain: biomedical Document type: Study Language: en As a potent oxidant, ozone has been demonstrated to inactivate SARS-CoV-2 through a multi-faceted mechanism. For instance, it can inflict damage on the viral envelope, a lipid membrane that safeguards the virus’s genetic material, which houses proteins vital for the virus to latch onto and infiltrate host cells, while ozone’s oxidizing properties can impair these proteins, thereby obstructing the virus’s ability to infect new cells . Furthermore, ozone can permeate the viral envelope to reach the viral genome, composed of RNA. By inducing breaks and mutations in the RNA, ozone incapacitates the virus’s ability to replicate or express its genes . The cumulative impact of ozone on the viral envelope and genome culminates in the inactivation of SARS-CoV-2 and other enveloped viruses, suggesting that atmospheric ozone could potentially serve as a natural deterrent against SARS-CoV-2 infection in pregnant women. Section title: Discussion Educational score: 4.208846092224121 Domain: biomedical Document type: Study Language: en Moreover, the application of ozone for COVID-19 therapy has shown promising results in various studies. A prospective case-control study demonstrated that ozone therapy was associated with lower mortality, shorter hospital stay, and improved oxygenation in patients with COVID-19 pneumonia . Furthermore, ozone has been found to be effective in inactivating airborne viruses, including a surrogate for SARS-CoV-2, in a wind tunnel simulating swine building, achieving more than 3 log10 reduction of viral infectivity within 6 minutes at concentrations of 0.5 to 1.8 ppm . Additionally, a study showed the feasibility of using ozone generated by a plasma device to disinfect face masks contaminated with SARS-CoV-2, where ozone at a concentration of 6 ppm could inactivate more than 99.9% of SARS-CoV-2 within 55 minutes . Consistently, we found protective effects of gestational ozone exposure on late pregnancy SARS-CoV-2 infection, which suggests that ambient ozone may directly participate in the clearance of SARS-CoV-2, or indirectly enhance the immunity of pregnant women and increase the resistance of relevant populations to SARS-CoV-2. Besides, Bernardino et al. proposes that ozone therapy could be used as an alternative or complementary therapy in the different phases of SARS-CoV-2 infection, depending on the dose and route of administration . They emphasize the rectal insufflation technique as a simple, safe, and inexpensive method of systemic ozone delivery. However, it calls for urgent experimental studies to confirm or rule out the biological effects of ozone therapy and to evaluate its safety and efficacy for the management of SARS-CoV-2 infection. Section title: Discussion Educational score: 4.072102069854736 Domain: biomedical Document type: Review Language: en Currently, ozone therapy has been explored for its potential to mitigate oxidative stress and exert antiviral effects, which could be especially beneficial during pregnancy. Studies have shown that ozone can reduce viral load, regulate oxidative stress, and balance the immune response, thus offering a potential therapeutic approach for managing infections like COVID-19 . While evidence is still emerging, ozone therapy’s effects on pregnant women require further investigation to ensure its safety and efficacy. In particular, the regulation of oxidative stress by ozone could be crucial in reducing the adverse outcomes associated with SARS-CoV-2 infections during pregnancy. Notably, ozone therapy has been used in adults for various, typically through methods like autohemotherapy, where controlled doses are administered . However, there is limited evidence regarding the safe use of ozone in neonates and pregnant women. The literature suggests caution in administering ozone therapy during pregnancy due to potential risks to both the mother and fetus. Controlled ozone administration may be a possibility for pregnant women, but further studies are needed to determine the appropriate dosages and safety protocols for this population. Section title: Discussion Educational score: 3.892277956008911 Domain: biomedical Document type: Other Language: en Although ozone therapy has shown promise as an antiviral treatment, it is important to note the potential risks associated with ozone exposure, particularly in high concentrations. Ozone is a potent oxidant that can cause respiratory irritation, damage to lung tissue, and other health issues when inhaled in large amounts . For pregnant women, the risks of ozone exposure are magnified due to the potential harm to both maternal and fetal health . Therefore, strict safety protocols and controlled ozone administration are essential to minimize these risks and ensure the safe use of ozone therapy in treating COVID-19 and other viral infections. Section title: Discussion Educational score: 4.0209527015686035 Domain: biomedical Document type: Study Language: en In the context of longitudinal ozone exposure over gestation and late pregnancy SARS-CoV-2 infection, both factors may affect the maternal immune system and fetal development in different ways. However, previous studies have also linked longitudinal ozone exposure over gestation to increased risks of preterm birth, low birth weight, intrauterine growth restriction, preeclampsia, and congenital anomalies. For example, late pregnancy SARS-CoV-2 infection is demonstrated to be associated with increased risks of severe maternal illness, preterm delivery, cesarean section, neonatal intensive care unit admission, and vertical transmission . Therefore, exploring the interaction between longitudinal ozone exposure over gestation and late pregnancy SARS-CoV-2 infection is crucial, which may contribute to the definition of an appropriate dose of gestational ozone exposure. Section title: Discussion Educational score: 3.9898841381073 Domain: biomedical Document type: Study Language: en Additionally, our findings suggest that higher levels of ozone exposure during pregnancy may reduce the risk of SARS-CoV-2 infection in late pregnancy. However, the applicability of these results to regions with different ozone levels remains uncertain. In areas with elevated ozone concentrations, there may be a different immune response to viral infections compared to regions with lower ozone levels. Further research is required to explore the impact of geographic variations in ozone exposure on COVID-19 susceptibility, particularly in populations with differing environmental conditions. Section title: Discussion Educational score: 4.100826263427734 Domain: biomedical Document type: Study Language: en There are several strengths and limitations of this study. The strengths include the robust sample size of 600 pregnant women, extensive data collection from electronic medical records, validated ozone exposure assessment based on hourly measurements, and adjustment for multiple confounders in the statistical analysis. However, there are some limitations that should be considered when interpreting the findings. First, the study was conducted at a single hospital in Jinan, China, which may limit the generalizability of the results. Second, ozone exposure was estimated based on ambient monitoring data rather than personal measurements, which may misclassify individual-level exposures. Third, the assessment of COVID-19 status was based on PCR testing at a single timepoint, which could underestimate the true prevalence of SARS-CoV-2 infection. Fourth, the retrospective design precludes establishing a temporal relationship between ozone exposure and COVID-19 onset. Finally, residual confounding by unmeasured factors cannot be ruled out. Overall, this study provides initial evidence on the association between gestational ozone exposure and SARS-CoV-2 infection, but additional longitudinal studies with more rigorous exposure assessment are warranted to confirm the findings. Section title: Conclusions Educational score: 4.08081579208374 Domain: biomedical Document type: Study Language: en To sum up, this retrospective cohort study found an inverse association between longitudinal ozone exposure over gestation and the risk of SARS-CoV-2 infection in late pregnancy among 600 pregnant women in Jinan, China. After adjusting for demographic and clinical factors, higher ozone exposure throughout pregnancy was associated with a significantly lower likelihood of testing positive for COVID-19 in the third trimester. However, this study is limited by its retrospective design and reliance on ambient ozone measurements, and hence further longitudinal investigations with more rigorous ozone exposure assessment at the individual level are warranted to validate the observed association between gestational ozone exposure and SARS-CoV-2 infection risk. | Other | biomedical | en | 0.999997 |
PMC11695409 | Section title: Introduction Educational score: 4.089727401733398 Domain: biomedical Document type: Study Language: en Patients with acute brain injury (ABI) exhibit gastrointestinal motility disorder ( 1 , 2 ). The autonomic nerve dysfunction resulting from ABI results in gastrointestinal muscle dysmotility, and the severity is associated with the intracranial pressure ( 3 ). On the other hand, ABI can activate the hypothalamic–pituitary–adrenal (HPA) axis and induce a systemic stress response that triggers intestinal smooth muscle inflammation, further leading to the disorder of intestinal smooth muscle contraction ( 4 ). Section title: Introduction Educational score: 3.8272745609283447 Domain: biomedical Document type: Study Language: en In patients with ABI, sedatives were indicated for controlling anxiety, pain, discomfort, agitation, facilitating mechanical ventilation, and also for “neuro-specific” indications such as reducing cerebral metabolic demands, and enhancing cerebral tolerance to ischemia ( 5 ). Sedatives are indispensable therapeutic components in therapeutic measures such as reducing intracranial pressure, maintaining temperature, and controlling seizure ( 5 ). However, the effects of sedation on gastric motility in patients with ABI have rarely been well studied. Section title: Introduction Educational score: 4.015511989593506 Domain: biomedical Document type: Study Language: en Gastric antrum ultrasound is an advanced technique developed in recent years ( 6 , 7 ). Antrum contraction can be observed to evaluate gastric motility directly. It is non-invasive and can be performed at the bedside. In this study, we used gastric antrum ultrasound to assess the effects of different sedatives on gastric motility in patients with ABI and compare their degree of inhibition of gastric antral contractions. Section title: Study design Educational score: 4.129161834716797 Domain: biomedical Document type: Study Language: en A prospective observed study was conducted. The sedative, either propofol (Group A), midazolam (Group B), or dexmedetomidine (Group C), was used according to the patients’s needs, and light sedation was applied. Gastric motility was measured with gastric antrum ultrasound before and after the administration of sedatives. The effects of different sedatives on gastric antrum contraction, which includes frequency (ACF), amplitude (ACA), and motility index (MI), were analyzed and compared. In addition, 18 adult healthy volunteers were recruited as normal controls to compare with patients with ABI. Section title: Study design Educational score: 0.9657252430915833 Domain: biomedical Document type: Other Language: en This research was approved by the Ethics Committee of Clinical Research and Experimental Animals of the First Affiliated Hospital of Sun Yat-sen University (Ethics No. 161). The institutional review board waived the requirement for informed consent since no intervention was performed and no personally identifiable information appeared. Section title: Patients recruitment Educational score: 1.7897837162017822 Domain: biomedical Document type: Study Language: en Patients hospitalized in the neurosurgery ICU of the First Affiliated Hospital, Sun Yat-sen University, from July 2018 to November 2018 were recruited . Section title: Patients recruitment Educational score: 2.8876235485076904 Domain: biomedical Document type: Study Language: en Inclusion criteria: 18–80 years old. Patients underwent ABI including intracranial hemorrhage, post-selective operation with brain tumors, or traumatic brain injury. Need therapy of short-acting sedatives like midazolam, propofol, or dexmedetomidine, and no oral hypnotic drugs were used. Section title: Patients recruitment Educational score: 2.167973041534424 Domain: biomedical Document type: Other Language: en Exclusion criteria: known upper gastrointestinal anatomical problems. Pregnancy. Section title: Sedation protocol Educational score: 2.369633436203003 Domain: biomedical Document type: Other Language: en The Richmond Agitation-Sedation Scale (RASS) was maintained at −2 ~ 1 points, meaning the depth of sedation was maintained at light sedation. A daily wake-up procedure was performed on each patient at 6–9 a.m. Section title: Gastric antrum ultrasound Educational score: 4.171641826629639 Domain: biomedical Document type: Study Language: en Gastric antrum ultrasound imaging was performed before and 20 min after sedation. All ultrasonic gastric motility monitoring in this study was completed between 9 and 10 a.m., which is 2 h later after the daily wake-up procedure. Enteral nutrition was performed at 60–80 mL/h through a nasogastric tube. Gastric antrum images were obtained with a 2.5–6 MHz curvilinear probe (SONIMAGE HS1 portable ultrasonic, Konica Minolta). Ultrasound examination was performed at 30-degree head-of-bed elevation and supine position. The antrum was located after identifying the liver’s left anterior lobe, the pancreas’s head, and the abdominal aorta. ACF was measured for 6 min, and an average number of gastric antrum contractions were observed every 2 min. The maximum gastric antral diastolic area (S max ) and minimum contraction area (S min ) were measured thrice. ACA was calculated as follows: ACA = (S max - S min )/ S max . MI was calculated as follows: MI = ACF × ACA. The same experienced sonographer performed all of the scans, as the force of ultrasound probe placement may affect the interpretation of the cross-section. Section title: Bowel sounds auscultation Educational score: 2.6605989933013916 Domain: biomedical Document type: Other Language: en Bowel sound auscultation was conducted before and 20 min after sedation. Bowel sound auscultation was for 3 min and the frequency of bowel sounds was calculated per minute. Section title: Other clinical data collection Educational score: 3.1498401165008545 Domain: biomedical Document type: Study Language: en The following clinical data were collected: age, gender, disease diagnosis, complications such as diabetes and coronary heart disease, body mass index (BMI), GCS score, and the utilization of basic medications such as proton pump inhibitors, or nonsteroidal anti-inflammatory drugs, or opioids, enrollment time after admission to ICU, length of ICU stay, and death. Section title: Statistical analysis Educational score: 3.4652907848358154 Domain: biomedical Document type: Study Language: en The SPSS software (version 23.0) was used for the statistical tests. Normally distributed data were expressed as mean ± standard deviation, non-normal distribution data median (interquartile ranges, IQR). The paired student’s test or the paired rank-sum test was adopted to analyze the difference before and after the sedative therapy. The least significant difference t -test (LSD-t) or Bonferroni method was applied for multiple comparisons. p < 0.05 was considered to be statistically significant. Section title: Baseline characteristics Educational score: 4.055721759796143 Domain: biomedical Document type: Study Language: en This study included 37 patients. Among them, 30 cases were with intracranial hemorrhage, 5 cases were with brain tumor, and 2 cases were with traumatic brain injury. In addition, 18 healthy volunteers were recruited for this study to compare gastric motility in patients with ABI. The sedative dosage was recorded. In this study, the dosage of propofol ( n = 13) was 0.036–0.043 mg/kg/h, the dosage of midazolam ( n = 12) was 0.031–0.067 mg/kg/h, and the dosage of dexmedetomidine ( n = 12) was 0.171–0.230 mg/kg/h. It is shown in Table 1 . Section title: Gastric motility in patients with ABI Educational score: 4.097762107849121 Domain: biomedical Document type: Study Language: en In this study, patients with ABI showed lower ACF (2.41 ± 0.89 times/2 min) and MI (1.25 ± 0.57) than health volunteers (ACF: 4.5 ± 0.39 times/2 min, p = 0.001; MI: 3.59 ± 0.24, p = 0.001). There was no significant inhibition of ACA (50.80 ± 11.60%), as shown in Table 2 . Section title: Influences of propofol on gastric antrum contraction and bowel sounds Educational score: 4.152858257293701 Domain: biomedical Document type: Study Language: en In patients receiving propofol ( n = 13), the ACA decreased significantly (53.31 ± 9.76% before vs. 26.41 ± 18.01% after, p = 0.002), ACF decreased significantly (2.27 ± 0.78 times/2 min before vs. 1.08 ± 0.74 times/2 min after, p = 0.002), and MI decreased significantly (1.14(0.59, 1.44) before vs. 0.84(0.09, 0.83) after, p = 0.002). The bowel sounds had no significant change after using propofol (3 (2.5, 3) times/min before vs. 3 (2, 3) times/min after, p = 0.317). It is shown in Table 3 . Section title: Influences of midazolam on the contraction of the gastric antrum and bowel sounds Educational score: 4.155062675476074 Domain: biomedical Document type: Study Language: en In patients receiving midazolam ( n = 12), the ACA decreased significantly (53.35 ± 13.87% before vs. 21.04 ± 12.47% after, p = 0.002), ACF decreased significantly (2.23 ± 1.04 times/2 min before vs. 1.32 ± 1.04 times/2 min after, p = 0.007), MI decreased significantly (1.48 (0.73, 1.62) before vs. 0.31 (0.04, 0.58) after, p = 0.007). The bowel sounds decreased after using midazolam (3 (2.25, 3) before vs. 2 (2, 3) after, p = 0.034). It is shown in Table 4 . Section title: Influences of dexmedetomidine on the contraction of the gastric antrum and bowel sounds Educational score: 4.160801887512207 Domain: biomedical Document type: Study Language: en In patients receiving dexmedetomidine ( n = 12), ACF decreased significantly (2.71 ± 0.86 times/2 min before vs. 1.85 ± 0.86 times/2 min after, p = 0.01), MI decreased significantly (1.37 (0.85, 1.5) before vs. 0.9 (0.35, 1.14) after, p = 0.01), and no significant difference in ACA (45.54 ± 9.98% before vs. 34.44 ± 16.04 after, p = 0.099) and bowel sounds (3 (2, 3) before vs. 3 (2.25, 3) after, p = 1). It is shown in Table 5 . Section title: Comparison of influences of midazolam, propofol, and dexmedetomidine on gastric antrum contraction Educational score: 4.130402565002441 Domain: biomedical Document type: Study Language: en Dexmedetomidine showed less inhibitory effects on ACA compared with midazolam and propofol. D-value of ACA was −11.10 ± 19.96% in dexmedetomidine, −32.21 ± 14.03% in midazolam, −26.90 ± 16.77% in propofol, p = 0.003. ACA suppression ratio was 20.67 ± 33.59% in dexmedetomidine, 60.43 ± 22.40% in midazolam, 51.50 ± 32.83% in propofol, p = 0.002 . Section title: Comparison of influences of midazolam, propofol, and dexmedetomidine on gastric antrum contraction Educational score: 4.140130996704102 Domain: biomedical Document type: Study Language: en Dexmedetomidine also showed less inhibitory effects on MI. MI suppression ratio was 36.00 ± 34.77% in dexmedetomidine, 68.81 ± 20.84% in midazolam, 60.69 ± 27.49% in propof ol, p = 0.012. D-value of MI was −0.28 ± 0.90 in dexmedetomidine, −0.89 ± 0.51 in midazolam, −0.4 ± 0.59 in propofol, p = 0.066 . Section title: Comparison of influences of midazolam, propofol, and dexmedetomidine on gastric antrum contraction Educational score: 4.144209861755371 Domain: biomedical Document type: Study Language: en There was no statistical difference in ACF. D-value of ACF was −1.08 ± 0.74 times/2 min in propofol, −0.96 ± 1.00 times/2 min in midazolam, 0.86 ± 0.94 times/2 min in dexmedetomidine, p = 0.811. ACF suppression ratio 49.74 ± 34.81% in propofol, 37.87 ± 35.24% in midazolam, and 29.0 ± 32.8% in dexmedetomidine, p = 0.518 . Section title: Discussion Educational score: 4.073590278625488 Domain: biomedical Document type: Study Language: en The clinical assessment of gastric motility has relied on clinical symptoms such as bloating and vomiting, which are subjective and untimely. Some studies used electrogastrogram to assess gastric motility and gastric emptying ( 8 , 9 ). However, the electrogastrogram can only represent gastric electrical activity and does not reflect effective gastric contraction. In this study, gastric motility was measured using bedside gastric antrum ultrasound. The gastric antral ultrasound can quantify the contraction amplitude and frequency of the antrum ( 10 ). Also, it is noninvasive, easy to operate, and can be performed at the bedside to observe the dynamic effect of clinical treatments. We found that ACF and MI deteriorated in patients with ABI. The finding is consistent with previous studies, which showed gastric motility disorders are severe ( 11 , 12 ). Section title: Light sedation has an influence on gastric motility in ABI patients Educational score: 3.661965847015381 Domain: biomedical Document type: Study Language: en Sedation is a common treatment for brain injury. Previous studies that explored the effects of sedation on gastric motility in patients with ABI have focused on deep sedation ( 13 , 14 ). However, light sedation is the current trend in ICU sedation management ( 5 , 15 ). This study found that light sedation can also reduce gastric motility in patients with ABI. Section title: Weaker gastric motility inhibitory effect of dexmedetomidine Educational score: 4.111055374145508 Domain: biomedical Document type: Study Language: en This study also compared the inhibitory effects of different sedatives on gastric motility. We found dexmedetomidine had less inhibitory effects on gastric motility than propofol and midazolam. Midazolam, a short-acting benzodiazepine, acts directly on the γ -aminobutyric ABId receptor ( 16 ). Propofol acts on the subtype A of the γ-aminobutyric ABId receptor ( 17 ). Different from propofol or midazolam, dexmedetomidine is a highly selective α 2 agonist, having mild sedative, mild analgesic, and antisympathetic properties ( 18 ). Dexmedetomidine showed a less inhibitory effect than midazolam and propofol, possibly due to its lighter sedation effect and sympathoexcitatory depressant function ( 19 ). In addition, dexmedetomidine has the ability to attenuate intestinal ischemia–reperfusion injury, inhibit the inflammatory response, and ameliorate the stress response ( 20 ). This could also rationalize the weaker inhibition of gastric antral contraction by dexmedetomidine. Section title: Midazolam caused a decrease in bowel sounds Educational score: 4.085117816925049 Domain: biomedical Document type: Study Language: en This study also observed bowel sounds to evaluate intestinal motility. We found that midazolam suppresses bowel sounds, but propofol and dexmedetomidine had no significant inhibition of bowel sounds. However, there are no studies to explore the effects of sedative medications on bowel sounds in patients with ABI. We conjecture that this might be related to the influence of sedatives on gastrointestinal hormones. Narchi et al. ( 21 ) found that sedatives, including midazolam and propofol, affect gastrin secretion. Early animal experiments revealed that midazolam intervention could cause a decrease in the secretion of motilin ( 22 , 23 ). Xu et al. ( 24 ) also found that propofol can increase the levels of gastrin and vasoactive intestinal peptide in patients undergoing gastrointestinal endoscopy. Moreover, in a clinical study of open surgery for colon cancer, patients undergoing intravenous anesthesia involving dexmedetomidine had a quicker recovery of gastrointestinal motility, and the levels of prokinetic gastrointestinal hormones such as motilin and gastrin in the plasma increased ( 25 ). Thus, It could be explained by the different mechanisms of midazolam, propofol, and dexmedetomidine affecting gastrointestinal motility and bowel sound. Section title: Limitations and strengths Educational score: 4.064004421234131 Domain: biomedical Document type: Study Language: en This is a prospective observational study. There are some limitations in this study. First, the clinical application of basic drugs such as opioids ( 12 ), proton pump inhibitors, and non-steroidal anti-inflammatory drugs ( 26 ) may influence gastric motility. However, the before-and-after comparison was employed in this study, which mitigated the errors brought by other therapies. Second, in this study, radioactive nuclides, gastrointestinal electrical examination, and capsule endoscopy were not utilized for the detection of gastrointestinal motility because these methods are not suitable for before-and-after comparison of short-acting sedatives. By contrast, gastric antrum ultrasound can be operated non-invasively and enables dynamic re-examination. Nevertheless, further studies were needed to explore the specific mechanisms of different sedatives affecting gastric motility. Section title: Conclusion Educational score: 3.871734619140625 Domain: biomedical Document type: Study Language: en Patients with ABI exhibited decreased gastric motility. All sedatives, either propofol, midazolam, or dexmedetomidine, had inhibited effects on gastric motilities. Dexmedetomidine has less inhibitory effects on ACA and MI compared with propofol and midazolam. | Study | biomedical | en | 0.999997 |
PMC11695411 | Section title: Introduction Educational score: 3.96012806892395 Domain: biomedical Document type: Review Language: en The field of genomics has rapidly evolved, significantly influencing clinical medicine ( 1 ). This evolution has led to advancements such as the diagnosis and understanding of rare and inherited diseases; guiding precision medicine in cancer treatment, and providing personalized risk assessment for disease development and treatment responses. However, despite widespread availability of genomic research findings, persistent socioeconomic and racial disparities exist in the diagnosis, treatment and usage of genomic medicine services ( 2 ). Inequitable access to genomic medicine and information, coupled with healthcare systems’ unpreparedness to offer genomic services universally, contribute to these disparities ( 1 , 3 ). Section title: Introduction Educational score: 4.1136794090271 Domain: biomedical Document type: Review Language: en Equity in genomic medicine is defined as the fair and equal application of genomic knowledge, ensuring everyone has access to services like testing and counselling, and that the implementation of genomic medicine is impartial ( 4 ). Researchers have argued that to address future equity in genomics we must first look at populations that have until now been left behind by the benefits of genomic medicine ( 4 ). There is substantial evidence to demonstrate that racial and ethnic minorities, Indigenous communities and rural residents have notably less access to genetics health services ( 5 , 6 ). For instance, a report by the United Nations highlighted the severe inequity faced by the Australian Aboriginal population in accessing health care ( 7 ). In Australia, reporting of Indigeneity is delayed, inconsistent and fragmented ( 8 ) which affects Indigenous Australians’ access to the health benefits and their representation in health data ( 39 ). Despite recognition of equitable healthcare as a fundamental human right, various social and health system barriers impede equitable access to genomic-integrated healthcare ( 9 , 41 ). Globally, diverse, marginalized and minority populations are under-represented at genomic health services, even though they have a higher prevalence of certain conditions and a strong demand for inclusion in clinical benefits ( 10 ). Hence, offering inclusive, accessible and universal services is crucial to extending these clinical benefits across all populations ( 11 , 12 ). Section title: Introduction Educational score: 3.2501213550567627 Domain: biomedical Document type: Study Language: en The field of genomic medicine is grappling with significant challenges in meeting its responsibilities to ensure equitable access to genomics. For instance, research examining equity in genomic health service use among Indigenous Australians suggests that there is a three-fold under-representation of Indigenous Australians, despite demand ( 13 ). This disparity is further evidenced by the Better Indigenous Genetic (BIG) Health Services study, which highlights profound inequities in both the provision and patient experience of clinical genetic services ( 6 ). Section title: Introduction Educational score: 2.708158493041992 Domain: biomedical Document type: Other Language: en The issue of inequitable access is not isolated but a global concern. Research from various regions shows that these disparities are widespread, and addressing them requires a fundamental redesign of healthcare systems. Increasing genomic awareness among people in lower socio-economic groups and culturally diverse and Indigenous populations is crucial. Strategies to enhance access to genomic services must be tailored to fit different contexts but are universally needed ( 14 , 21 ). Section title: Introduction Educational score: 2.7855522632598877 Domain: biomedical Document type: Review Language: en This is the first evaluation of genomic policy that identifies the level of inclusion of the concept of equity and its shortfalls. This policy review focuses on the concept of equity in access to genomic care, the level of inclusion of equity and how it is addressed and what mechanisms are in place to achieve equity in genomic care in the international health policy. Section title: Health equity and right for health Educational score: 3.8620972633361816 Domain: biomedical Document type: Review Language: en Paul Farmer’s words denote the intrinsic connection between the idea of the right to health and health equity. Human rights and health equity are profoundly linked to each other ( 15 ). His words also indirectly encapsulate the notion of health equity as a challenging exercise and the inadequacy of the current global health systems to achieve it. While demonstrating that both human rights and equity strive for the same aim--equal opportunity--Braveman and Gruskin further argue that the concept of human rights gives a universally applicable framework for the attainment of health equity . Recent genomic discoveries and research have contributed to improved health outcomes and reduced morbidity and mortality. However, benefits of genomic discoveries are not distributed equally to all groups in a population. There are multiple socio-economic, geographical and cultural barriers that exist for equal provision and access to genomic health care in different contexts and this policy analysis explores how equity is embedded in global genomic health policy. In general, achieving equity has proved to be an evasive objective in public policy not only in health but also in other areas such as education ( 16 ) and public administration ( 17 ). Section title: Health equity and right for health Educational score: 3.858301877975464 Domain: biomedical Document type: Review Language: en Equity is a key principle of the concept of “health for all” by United Nations that was brought forward by the Alma Ata Declaration ( 47 ) four decades ago. WHO defines health equity as ‘the absence of unfair, avoidable, remediable differences among groups of people’ ( 40 ) even though differences exist between the groups in the form of gender, sexual orientation, ethnicity, wealth, residence/ geographic location. Equity has been defined as having equal access to available care for equal need, equal utilization of services for equal need and equal quality of care for all [( 18 ), p. 434]. In an attempt to define the meaning of equity in the health arena, in the 90s Whitehead affirms that equity aligns with seven principles: (i) Equity policies must focus on improving living and working conditions; (ii) Equity policies must be directed towards enabling healthier lifestyle; (iii) Equity policy must focus on engaging community participation and decentralization of decision making power; (iv) Equity policies must assess the impact on health by policies in all other sectors, especially the impact on health of Vulnerable Groups; (v) Equity policies must have mutual concern and control at the international level; (vi) Equity policies must assure universal access to high quality health care; and (vii) Equity policies should be based on relevant research, monitoring and evaluation ( 18 ). Section title: Health equity and right for health Educational score: 3.108161687850952 Domain: other Document type: Study Language: en Two decades after the publication of these health equity principles, policy researchers show the importance of narrowing down these broad principles and affirm the importance of making a distinct and explicit commitment to achieve equity in health with specific objectives. Mannan et al. ( 19 ) argue that governments’ action must be pro-equity. Drawing from EQUINET, they affirm that equity in health policy can be referred to as a “propitious political message” (p. 7) that is intended to bring social cohesion and solidarity through concrete political steps to provide mechanisms to protect the health of the poor. Equity is a cornerstone of policy making and its achievement depends on what the decision makers consider as priority health issues and the selection and inclusion of populations that most deserve attention ( 19 ). We delve into the details of how political leadership becomes a main driving force of achieving health equity in our next publication that focuses on the experiences and perceptions of achieving equity in genomic health of the policy makers in the international contexts. Section title: EquiFrame to assess equity in policy Educational score: 3.563755750656128 Domain: biomedical Document type: Review Language: en In the development of EquiFrame, Mannan et al. ( 19 ) drew heavily from Braveman and Guskin’s advocacy for the routine and systematic application of equity and human rights approach to health sector actions . They also state that the human rights approach facilitates the analysis of policy from a range of diverse perspectives. The EquiFrame was developed using perspectives from human rights, the right to health and vulnerability. This framework facilitates the exploration of the inclusion of equity in genomic policy and it gives us a clear view of the level of commitment these policies employ to achieve health equity. Mannan et al. make reference to 21 Core Concepts (CCs) that are focused on the individual and the collective in relation to “principles of universal, equitable and accessible health services” [( 19 ), p.13]. Section title: Contextualizing genomic health policy Educational score: 3.802903413772583 Domain: biomedical Document type: Review Language: en In general, policy is influenced by factors such as understanding and framing of health; use of evidence; contextual priorities and political ideologies; systems; and leadership ( 20 ). Inequity in access to genetic health care among diverse populations has been attributed to inadequate integration of genomics policies into the general health systems and policy gaps in genomic health itself ( 21 , 46 ). These policy challenges need to be addressed through better understanding of the barriers for integration of genomics into policies, through policy frameworks that are informed by population health priorities, and through engagement with underserved communities, health care providers and policy makers ( 21 , 22 , 45 , 46 ). Even in many parts of the developed world, it appears that genomic policy has not caught up with the developments of genomic research and its benefits. Section title: Contextualizing genomic health policy Educational score: 4.005075454711914 Domain: biomedical Document type: Review Language: en Khoury et al. ( 5 ) state that an agenda of health equity in genomics needs to go beyond clinical research and should include health policy. The authors argue that what they term as a public health agenda is needed to address the disparities in implementation of genomics in different populations. To address these disparities, they state that this public health action needs to focus on, (1) population-specific needs and outcomes assessment, (2) policy and evidence development and (3) assurance of delivery of ethical and effective interventions. They further argue that absence of concerted public health action and further advancements of genomics will only widen the equity gap. The current review that explores the inclusion of equity concepts in existing genomic health policy and identify barriers and challenges to equity-informed genomic policy, aims to support the type of public health agenda that Khoury et al. ( 5 ) advocate for. Section title: Concept of equity Educational score: 3.430140733718872 Domain: biomedical Document type: Other Language: en Equity in genomics has been defined as the successful implementation of genomic medicine where all populations have equitable access to effective affordable genomic medicine which includes diagnosis, treatment and prevention strategies ( 4 ). Genomic health policy has a crucial role to play in ensuring the equitable distribution of and access to genomic medicine. As there are currently many efforts being made to translate genomic medicine benefits into health care services, it is an important time for policymakers along with researchers, funders and administrators to capitalize on this progress to ensure equitable distribution of health benefits ( 4 ). Section title: Concept of equity Educational score: 2.7536628246307373 Domain: biomedical Document type: Study Language: en In such an exercise, first it is important to understand whether and how equity is integrated into genomic policy and what strategies policymakers have already implemented to ensure equity. In this policy analysis we will use the EquiFrame framework designed by Mannan et al. ( 19 ) to rate and analyze the level of incorporation of the concepts of inclusion and equity in genetic health policies internationally. Section title: Objectives Educational score: 3.862138271331787 Domain: biomedical Document type: Review Language: en The aim of this policy review is to identify approaches that will support the development of policy at a health system level to enable the equitable distribution of benefits of genomic health services to marginalized populations including First Nations, as the field continues to develop. To do this, the policy analysis explored the level of alignment with concepts of equity in global genomic policies in relation to making genomic testing and medical benefits accessible to everyone and identified exemplars of equitable policy approaches to genomics services. Key factors that contribute to equitable genomic services in global genomic policies are identified and recommendations provided for equitable genomic health policy development; both in relation to the content of equitable genomic health policy and with regards to equitable processes in policy development. Section title: Objectives Educational score: 1.1682173013687134 Domain: other Document type: Other Language: en In this policy analysis we will explore the following research questions. Section title: Methods Educational score: 1.8855438232421875 Domain: biomedical Document type: Study Language: en The first step of conducting the policy analysis was the search for active genomic health policies internationally. The below table shows the inclusion and exclusion criteria ( Table 1 ). Section title: Methods Educational score: 2.1909375190734863 Domain: biomedical Document type: Study Language: en Three databases were used (Medline Ovid, Scopus and CABI Global Health) for the search and in addition General and Specific Policy Repositories (Genomic Medicine Policy), Global Consortia in Genomic Medicine, WHO Collaborating Centres in Genomics, Australian Genomics, Public Policy Projects, Global Genomic Medicine Consortium (G2MC), G2MC conference Oct 2023 and National Human Genome Research Institute (Database on genetic policy and laws) databases were searched. Section title: Methods Educational score: 2.3122775554656982 Domain: biomedical Document type: Other Language: en The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) diagram below demonstrates the process we followed in searching and selecting the policies 1 . Section title: Methods Educational score: 3.615330934524536 Domain: biomedical Document type: Review Language: en We reviewed 17 genomic policies in Europe, Southeast Asia, Canada and Australia. Policy is defined by Howlett and Cashore ( 23 ) as the “actions that contains goals and the means to achieve them, however well or poorly identified, justified, articulated and formulated” (p. 17) while Dye ( 42 ) sees it simply as the actions that a government decides to take and not take. We have employed EquiFrame to analyze the extent to which policies in genomics have employed the concepts of equity, social inclusion and human rights ( 19 ). EquiFrame has been developed by Mannan et al. ( 19 ) as a framework that can be used to assess the degree to which equity has been addressed in policies and policy related documents. EquiFrame views social inclusion and human rights as key components of equity. Furthermore, EquiFrame enables the evaluation of the degree to which a health policy demonstrates commitment to 21 CCs of human rights and to 12 Vulnerable Groups (VGs), guided by the ethos of universal, equitable and accessible health services. According to the EquiFrame authors, this framework can be customised to the requirements of the purpose of the analysis ( 19 ). Section title: Methods Educational score: 2.19765305519104 Domain: biomedical Document type: Other Language: en Accordingly, the EquiFrame framework (a) defines Core Concepts, (b) identifies the key questions and key language on which the concept is based, (c) identifies Vulnerable Groups, and (d) provides a data extraction matrix to chart the analyzed documents. The EquiFrame Matrix lists the 21 Core Concepts along the vertical axis, and 12 Vulnerable Groups along the horizontal axis. Section title: Methods Educational score: 1.0837026834487915 Domain: other Document type: Other Language: en For instance, VGs and CCs may be added or removed to suit specific requirements, political, cultural or other contextual interests or constraints. In this analysis, Indigenous groups are added to the VGs which makes the total of 13 Vulnerable Groups that each selected policy is assessed. Section title: Methods Educational score: 1.3393393754959106 Domain: other Document type: Other Language: en Accordingly, the matrix was developed with the 21 CCs along the vertical axis and 13 VGs along the horizontal axis for each of the selected policy document. Section title: Scoring Educational score: 1.2234373092651367 Domain: other Document type: Other Language: en Each policy was assessed against the 21 Core Concepts of the EquiFrame framework and received a score from 1 to 4 for each Core Concept. This is a rating of the quality of commitment to the Core Concept within the policy document: Section title: Scoring Educational score: 1.3652845621109009 Domain: biomedical Document type: Other Language: en 1 = Core Concept mentioned. Section title: Scoring Educational score: 1.2902497053146362 Domain: biomedical Document type: Other Language: en 2 = Core Concept mentioned and explained. Section title: Scoring Educational score: 1.1953723430633545 Domain: other Document type: Other Language: en 3 = Specific policy actions identified to address the Core Concept. Section title: Scoring Educational score: 1.3990403413772583 Domain: other Document type: Other Language: en 4 = Intention to monitor concept was expressed. Section title: Scoring Educational score: 1.1023247241973877 Domain: other Document type: Other Language: en If a Core Concept was not relevant to the document context, it is stated as not applicable. Section title: Scoring Educational score: 1.3152662515640259 Domain: other Document type: Other Language: en In each document, the presence of Core Concepts is addressed for each Vulnerable Group that is identified in the policy. If no Vulnerable Group was mentioned, but a Core Concept addressed the total population (e.g., “all people”), the Core Concept was scored as Universal. The total number and scores mentioned in the Core Concepts and Vulnerable Groups is calculated for each document, across all countries the policies originate from. Section title: Scoring Educational score: 2.191215753555298 Domain: biomedical Document type: Study Language: en Inter-rater reliability was achieved through comparing separate evaluations between two raters. Two raters independently reviewed the EquiFrame analysis. Regarding inter-rater reliability for the application of EquiFrame to identified policies, percentage agreement was calculated regarding all summary indices for each policy. Section title: Summary indices for EquiFrame Educational score: 1.1637250185012817 Domain: other Document type: Other Language: en Four Summary Indices of EquiFrame are outlined below. Each policy is rated according to the percentage they received for addressing and the quality of the Core Concepts and Vulnerable Groups. Section title: Results Educational score: 1.48868727684021 Domain: biomedical Document type: Other Language: en A total of 17 policies were selected from the desktop search of literature. The policies came from Australia, Hong Kong, Thailand, India, United Kingdom, Denmark, Finland, Italy and Canada. One of the 17 policies is an international genomic policy developed by the International Rare Disease Consortium. Section title: Results Educational score: 1.3066980838775635 Domain: other Document type: Other Language: en Out of the 17 policies, most rated reasonably high (around or above 50%) for the Core Concept coverage. A smaller number of policies scored reasonably for the Vulnerable Group coverage. Indigenous populations, people from culturally and linguistically diverse backgrounds and people living remotely were the three main Vulnerable Groups addressed. Few policies scored reasonably for the Core Concept Quality, which was calculated from the categories of either stating a specific policy action, or intention to monitor action in the policy. Section title: Inclusion of core concepts Educational score: 0.9832823276519775 Domain: other Document type: Other Language: en In this section only the concepts that are most frequently addressed across the 17 policies are reported and summarized. In each section, a comment is made on the Core Concept’s EquiFrame analysis rating, with a summary of how the Core Concepts are included in the policies and an attempt to compare policies. Section title: Access Educational score: 1.1284074783325195 Domain: other Document type: Other Language: en The Core Concept of ‘access’ is the most widely addressed concept related to equity in the selected policies. Access was addressed in 14 out of 17 policies (82%). The ratings that are given in the EquiFrame for the Core Concept of access in the 14 policies varies between 1 and 2 (between ‘access’ being stated in the policy to ‘access’ being explained). Section title: Access Educational score: 1.4745861291885376 Domain: other Document type: Other Language: en Out of these 14 policies, the genomic policies of Western Australia , New South Wales Genomics Strategy and in the United Kingdom, one of rare diseases and the National Health Services genomic policy mention the necessity of equitable access to genomic medicine in varying degrees. Section title: Multi dimensionality and priority areas of access Educational score: 2.084287643432617 Domain: biomedical Document type: Other Language: en Multi dimensionality of access is an aspect that is acknowledged in most of these policies. For example, the Australia National Health Genomic Policy Framework 2018–2021 details the multidimensionality of access as it includes factors such as location, cost, availability and cultural acceptability which would drive the ability to access genomic care especially of vulnerable populations. For example, the policy identifies the dimension of culturally secure, appropriate and responsive genomic services as a priority in relation to addressing the problems of access to genomic services of the Aboriginal and Torres Strait Islander populations. Section title: Multi dimensionality and priority areas of access Educational score: 1.6810815334320068 Domain: biomedical Document type: Other Language: en Under Western Australia Genomic Health Policy , ‘access’ is also referred to as the health consumers’ ability to access educational material and increased awareness of the applications of genomic innovations in order to receive benefits from genomic healthcare. Section title: Multi dimensionality and priority areas of access Educational score: 1.6345998048782349 Domain: other Document type: Other Language: en In some policies there are specific priority areas that are identified related to access. Both in the WA policy and the UK Rare diseases Framework, the use of novel and advanced technology and digital tools is referred to as a priority to increase remote access for patients in WA and the UK. According to the two policies, improving access to specialist care and treatment entails innovation and commitment for innovation and collaboration of the health system with stakeholders. Section title: Multi dimensionality and priority areas of access Educational score: 2.0357134342193604 Domain: other Document type: Other Language: en Providing access to vulnerable populations is also identified as a strategic priority in some policies. WA policy recognizes that Indigenous populations, people from culturally and linguistically diverse backgrounds and communities living in rural and remote regions need to be given priority when championing access to value based genomic services. In addition, the Australia National Health Genomics Policy Framework 2018–2021 too identifies the principle of equity in access as an area of strategic priority specifically related to vulnerable populations (p. 4). The UK Rare Diseases Framework includes the Core Concept of access mainly in relation to Black, ethnic minority communities and patients living in remote parts of the country. Section title: Increasing access through the mainstream health care Educational score: 1.3797935247421265 Domain: other Document type: Other Language: en Both the policies of WA and NHS in the UK recommend the integration of genomic services to the mainstream health system to reinforce equitable access of genomic services. Section title: Increasing access through the mainstream health care Educational score: 2.445540189743042 Domain: biomedical Document type: Other Language: en For example, the vision of the UK genomic policy Accelerating Genomic Medicine in the NHS ( 43 ), is that treatment is accessible to all, as part of routine care at NHS. This policy sets out to achieve the vision through four priority areas; (1) by embedding genomic testing and medicine across NHS (2) by providing equitable genetic services for improved outcomes (3) by ensuring genomic data could be interpreted and used by other diagnostic data (4) by evolving the service through cutting edge science (so that the patient can make use of rapidly evolving improvements in the science). Section title: Increasing access through the mainstream health care Educational score: 1.862716555595398 Domain: other Document type: Other Language: en The WA policy also identifies that integration of genomic care in the mainstream health care is an important strategy to increase access for the growing demand for genomic health care. Ensuring that WA health systems have processes, expertise and infrastructure in place to evaluate and implement continuous advances in genomic health services in the mainstream health care are identified as important strategies to increase access to genomic health services. Section title: Challenges to access Educational score: 1.704613447189331 Domain: other Document type: Other Language: en Some policies acknowledge challenges specifically related to access to genomic health care. For example, the WA policy recognizes continuing mistrust within Indigenous communities towards the healthcare system and genetic testing due to a historical lack of transparency and culturally appropriate ways of obtaining consent and culturally inappropriate uses of genomic data as a barrier to access. Section title: Challenges to access Educational score: 1.7678325176239014 Domain: biomedical Document type: Other Language: en The UK Rare Diseases Framework identifies the availability of limited data as a challenge to providing equitable access to genomic health services to patients with rare diseases. Another challenge according to the policy NHS policy is to find the balance between the provision of treatment for all patients with the need and the limited and fixed resources (p. 15). Section title: Challenges to access Educational score: 1.3675025701522827 Domain: other Document type: Other Language: en As a solution to address some of these challenges, WA policy points to the importance of the health system being cognizant of the need for cross sector training, educational requirements and working arrangements across private, public and not for profit genomic service sectors to reinforce equitable access. Section title: Participation Educational score: 3.0053958892822266 Domain: biomedical Document type: Review Language: en The Core Concept ‘participation’ was addressed in genomic policies of Australia, UK, Canada, Hong Kong and India to varying degrees. Twelve out of the 17 genomic policies (70.5%) included participation as a Core Concept. Participation in them is addressed in two distinct areas: participation of the community in genomic research and community participation in genomic policy development and implementation. Participation of the community in genomic research is not in the scope of this review. In the latter category, the key question posed regarding the Core Concept of participation in the EquiFrame is if the policy supports the right of vulnerable groups to participate in the decisions that affect their lives and enhances their empowerment. The ratings of the 12 policies vary between 1 and 3 (from naming the Core Concept to detailing methods to achieve the Core Concept). Section title: Participation of vulnerable groups Educational score: 1.5533573627471924 Domain: biomedical Document type: Other Language: en UK policies of Rare Diseases Framework and the ‘Accelerating Genomic Medicine in the NHS’ ( 43 ) seem to take lead in acknowledging the importance of participation of vulnerable populations in the development of genomic policy. Section title: Participation of vulnerable groups Educational score: 2.214836359024048 Domain: biomedical Document type: Other Language: en A UK genomic policy that gives a high degree of attention to patient and community participation and collaboration is the Rare Diseases Framework. Although participation is not mentioned explicitly, patient and community voices and lived experience are an underpinning theme to achieve the framework’s four key priorities. It acknowledges that patients’ voice is at the center of decision-making in relation to all aspects in genetic care and service delivery, including policymaking. It focuses on the inclusion of patient representatives from Black, ethnic minority and disadvantaged communities (p. 17). Section title: Participation of vulnerable groups Educational score: 3.4352357387542725 Domain: biomedical Document type: Other Language: en The UK policy ‘Accelerating Genomic Medicine in the NHS’ ( 43 ) embeds patient and public participation as a core principle and a key determinant in the design and of the success of the NHS Genomic Medicine Service (GMS) (p.15). The policy describes a strategy of achieving participation through multiple avenues and includes separate sets of recommendations to health providers, healthcare professionals, patients and patient groups to increase participation in genomic healthcare and research at all levels of NHS GMS. The role of health providers under the policy includes identifying unmet needs and inequalities and providing access for patients under their care to participate in genomic clinical trials and projects as well as genetic testing offered by NHS. The policy also supports increased patient and patient groups’ involvement in NHS GMS national and regional governance structures at all levels and promotes the role of the NHS in supporting such involvement. Thus, participation of patients and patient groups is included in this policy as an action to be taken in the development of governance policies. Section title: Participation of vulnerable groups Educational score: 2.7652366161346436 Domain: biomedical Document type: Other Language: en Further, even though the UK genomic policy, Genome UK: the Future of Healthcare, refers to the Core Concept of participation only in relation to genomic research, it acknowledges an important barrier for the diversity of genomic datasets that exists in racially different communities. Participation in the policy is emphasized as an important step to achieve genomic equity of access and diversity (p. 39). The policy recognizes that the distrust and suspicion of Black Caribbean and ethnic minority communities towards genomic research and the assumptions of health professionals about ethnic minority population are possible barriers for participation. Section title: Participation of vulnerable groups Educational score: 1.9899101257324219 Domain: biomedical Document type: Other Language: en The Canadian genomic policy too, focuses on participation of vulnerable populations in developing genomic policy only to a limited extent. Strategic Plan 2022–2027: Sequencing our future: A Vision for a Healthier Future ( 44 ) outlines four commitments: (1) enabling genomic medicine; (2) improving genetic disease diagnosis and therapies; (3) embracing diversity, inclusion and Indigenous rights; and (4) strengthening the community (capacity building). Participation and co-design are only mentioned in relation to genetic research under commitment 1 (p.11). Participation in clinical genetic services is not the focus here. Section title: Participation of consumers in policy implementation Educational score: 1.2770164012908936 Domain: other Document type: Other Language: en Participation in the Australian genomic policies are not specifically mentioned in relation to the engagement of any specific vulnerable population. Section title: Participation of consumers in policy implementation Educational score: 2.9818062782287598 Domain: biomedical Document type: Other Language: en Within Australian genomic policies, in the NSW Health Genomic Strategy: Implementation Plan 2021–2025, participation of consumers is incorporated in the implementation activities of the policy. Rather than consumers participating to co-develop the policy, it consolidates key actions that need to be taken to achieve the genomic strategy through a participatory and co-design approach with the key stakeholders. The key actions proposed in the strategy which incorporates the participation of the consumers are embedding, scaling and sustaining multidisciplinary clinical genomic models of care; developing and testing tools to support triage and referral pathways and to develop tools to translate genomic research into clinical practice appropriately and consistently; continuing to monitor requirements for data management, governance and access levels; developing guide to standards of genomic products that incorporates consumer needs and experiences; developing an electronic genomic tracking system; implement and integrate test result reporting with existing systems; establishing digital consent requirements for genomic testing; build and test digital consent for clinical genomic use; integrating patient and health professional educational resources with digital consent; developing educational website design to meet consumer requirements; redesign the educational resources portal; developing training needs assessment tools for workforce; and using workforce and educator champions support to deliver clinical genomics use . Section title: Participation of consumers in policy implementation Educational score: 1.9745954275131226 Domain: other Document type: Other Language: en Within the Australia National Health Genomics Policy Framework 2018–2021, the terms ‘engagement’ and ‘participation’ are used interchangeably. The Framework recognizes the importance of consumer participation in effective health care delivery and conceptualizes individuals and families as active and engaged partners with the genomics health care delivery team. The policy framework also identifies stakeholder engagement as a key enabler of implementation success. The ‘Genetic and Genomic Healthcare for Victoria 2021: Improving the Health and Wellbeing of Victorians’ policy also integrates the Core Concept of participation in a universal sense. Section title: Participation of consumers in policy implementation Educational score: 1.3874505758285522 Domain: other Document type: Other Language: en ‘Bringing innovation to life: Strategic Vision’ outlines the mission and vision of Genome Canada. Even though this is primarily relevant to researchers and research processes under Genome Canada, the policy also discusses service delivery to a limited extent. The Core Concept of participation and engagement is described as concepts through: Section title: Integration Educational score: 3.4856693744659424 Domain: biomedical Document type: Study Language: en In the EquiFrame manual the Core Concept of integration means integrating genomic care for Vulnerable Groups in the main or general healthcare system rather than having specialized services dedicated for Vulnerable Groups. Mannan et al. ( 19 ) argue that when incorporating the concept of integration meaningfully in policy, one must first investigate if the policy bars the vulnerable communities in participating in mainstream healthcare. The authors further argue that dedicated services for vulnerable populations could have inadvertent effects such as stigmatization, be at the risk of losing funds, become less desirable for health professionals and therefore face the risk of losing quality of care (p.54). This analysis demonstrates that the CC of integration in relation to including vulnerable population. Section title: Integration Educational score: 2.3767106533050537 Domain: biomedical Document type: Review Language: en Overall, out of the total number of 17, 12 policies in the review either did not focus on integration or used the CC of integration in a manner that did not align with the meaning of the CC as outlined by Mannan et al. ( 19 ). The term ‘integration’ in these policies is used to denote the integration of genomic data and research in the genomic services; this is not within the scope of this review. Section title: Integration Educational score: 2.081026077270508 Domain: biomedical Document type: Other Language: en Five policies from Australia, UK and Denmark address the CC of integration of genomic services in the mainstream general healthcare services in varying degrees and detail. What these five policies demonstrate in common is the importance of integrating genomic clinical services in the general healthcare services in the delivery of person centered, equitable genomic care. Only two policies specifically address integration in the healthcare system with the intention of addressing the needs of vulnerable populations. While the policies from the UK emphasize the strong platform that NHS provides the Genomic Medicine Service (GMS) in the delivery of equitable, effective and sustainable genomic healthcare for the UK population, Australian genomic policy clearly demonstrate its commitment to integrate genomic services into general healthcare services. Section title: Integration Educational score: 2.2256815433502197 Domain: biomedical Document type: Other Language: en Accelerating Genomic Medicine in the NHS recognizes that NHS is in a strategic position to implement genomic healthcare as a nationally organized locally delivered service. The policy points out that Genomic Medicine Service (GMS) of NHS is an integrated service model which have brought together genomic testing, clinical genetics and other services, research and innovation to deliver the benefits of genomics to all NHS patients (p. 16). The NHS policy states that the development of an integrated service model of genomic service via primary and community care is specifically focused at providing services for unmet needs and undiagnosed populations. Section title: Integration Educational score: 1.4143953323364258 Domain: other Document type: Other Language: en The UK Rare Diseases Framework too echoes this statement of how Genomic Medicine Service is integrated into the frontline healthcare service of NHS. Section title: Integration Educational score: 1.8282784223556519 Domain: other Document type: Other Language: en Australia’s National Health Genomics Policy Framework focuses on the CC of integration in relation to integrating genomic healthcare in the national health system. The policy also acknowledges that the integration of the genomic services in the national health system is dependent on the acceptance of this by the community and the level of confidence that the community has on the genomics (p. 1). Even though it does not mention vulnerable populations in relation to integration, the policy views integration of genomic services in the national health system as a mechanism to address inequity and also recognizes the risk it still presents for potential discrimination of people. Section title: Quality Educational score: 1.1739401817321777 Domain: other Document type: Other Language: en The Core Concept of quality has been recorded in 10 policies (58%) from Australia, Canada, Thailand, Hong Kong, Italy and Denmark. In most of these policies, ‘quality’ was the highest rated Core Concept that was discussed about in a universal sense. Section title: The features and definitions of quality Educational score: 2.4455535411834717 Domain: biomedical Document type: Review Language: en Adherence to quality and safety when promoting public trust in genomics in health care is considered to be the main underlying principle in the WA genomic strategy. Genomic policies around the world characterize quality in different ways. Quality referred to in genetic tests and services are both included in this review as they are both intrinsically related. Section title: The features and definitions of quality Educational score: 1.8785138130187988 Domain: biomedical Document type: Other Language: en One common trend we see across these policies is that achieving high quality in genomic care is seen to be obtainable through a person-centered approach and highly skilled healthcare workforce. Section title: The features and definitions of quality Educational score: 2.266092538833618 Domain: biomedical Document type: Other Language: en For example, the WA genomics strategy characterizes health care quality as the result of a person and family centered approach along with better health outcomes, improved safety, cost effectiveness and consumer satisfaction (p.18). Section title: The features and definitions of quality Educational score: 1.865348219871521 Domain: biomedical Document type: Other Language: en High quality genetic healthcare service in the Genetic and Genomic Healthcare in Victoria is characterized by a skilled healthcare workforce that is able to build trust with the public and raise awareness of benefits and limitations of genomic care and on the growth and development of knowledge in the area (p. 12). Section title: The features and definitions of quality Educational score: 1.786985993385315 Domain: biomedical Document type: Other Language: en Proven high quality clinical services, safety, clinical utility and effective safe use of genomic data are related to quality in the genomic policy ‘Strategic Development of Genomic Medicine in Hong Kong (p. 39). Quality and efficiency are addressed specifically in relation to genetic tests and genetic services. Section title: The ways to achieve quality Educational score: 1.786102533340454 Domain: biomedical Document type: Other Language: en The selected policies name strategies such as strengthening the overall health system, implementing high-quality genetic testing and implementing quality assurance mechanisms in all components of genomic services to improve quality in specific countries. For example, the Genetic and Genomic Healthcare in Victoria views strengthening the healthcare system as a key component of quality service provision. Section title: The ways to achieve quality Educational score: 1.7781535387039185 Domain: biomedical Document type: Other Language: en In the Danish genomic policy 2020–2021, the country’s ability to provide high-quality genetic analysis to all patients irrespective of where they live is the outcome of continuous work on personalized medicine in Denmark, its consolidation and streamlining of clinical activities and supportive infrastructure (p. 8). Section title: The ways to achieve quality Educational score: 1.7088652849197388 Domain: biomedical Document type: Other Language: en In the policy Genome British Columbia , quality is mentioned as a requirement for successful clinical implementation (p. 14). The policy recommends the implementation of quality assurance mechanisms to ensure clinical and laboratory infrastructure and capabilities to perform genome analysis appropriately (p. 15). Section title: The ways to achieve quality Educational score: 1.7233598232269287 Domain: biomedical Document type: Other Language: en In Thailand’s National Biotechnology Policy Framework , quality healthcare is discussed in relation to equity in access. Quality is addressed in a broad manner and is not only directed to clinical genomic services. Overall, improvement of quality of life is a main target of the policy through biotechnology. In the Thai genomic policy, there are four sectors: food and agriculture, medicine and health, bio energy and bio-based industry, and quality is prioritized in all four sectors. Section title: Coordination of services Educational score: 3.7952640056610107 Domain: biomedical Document type: Study Language: en The Core Concept ‘Coordination of services’ is addressed to a moderate extent in the selected policies. Mannan et al. view coordination of services as the ability of health professionals to deliver services through the established interpersonal relations, through structural mechanisms that liaise different agencies that connect different levels of local, state and federal governments in different health care systems (p. 48). Coordination of services was addressed only in 8 policies (44%) from Australia, UK, Hong Kong, Finland and Canada. The EquiFrame ratings for these policies vary from 1 to 4, the highest rating that can be given to a policy for detailing specific policy actions to achieve the Core Concept and methods to monitor the progress of the specified policy actions. While some policies acknowledge the important role that the state government or the national health services/system must play in coordinating the services (Such as the WA genomic strategy and Accelerating Genomic Policy in the NHS), others identify the ways that this can be achieved (Such as the UK Rare Disease Framework) and how integration of clinical genetic care can into routine healthcare can be achieved through coordination of services . None of the policies referred to any specific vulnerable groups in relation to coordination of care. Section title: Coordination of services Educational score: 2.104442834854126 Domain: other Document type: Other Language: en The policy that earned the highest rating for coordination of services is the WA genomic strategy . The policy views the state government as the entity that would coordinate between different agencies and organizations to achieve the type of genomic care that is explained in the policy (p.10). In this policy, under Priority 1 (a person- and family-centered approach to genomic healthcare), a coordinated and personalized approach is prioritized to meet the needs, values and preferences of the consumer and the family (p. 18). In Priority 2 (genomic health services), the policy emphasizes the importance of collaborative and coordinated mechanisms in health service planning and capacity-building to integrate new genomic advancements into the WA health system (p. 23). The policy further explains this integration through the example of appropriate coordination between state-wide pathology and clinical genomic services and other healthcare providers to provide equitable and ethical genomic care to the WA population. The policy states that strong stewardship, leadership and governance are required to achieve service coordination to deliver the strategy (p. 40). Section title: Coordination of services Educational score: 2.0777041912078857 Domain: other Document type: Other Language: en Coordination of care is a policy priority in the UK Rare Diseases Framework . The policy identifies the importance of coordination of various services for rare disease management and the challenges that exist for the coordination of this care (p.14). According to the policy, care coordination helps minimize burden on patients and their carers and helps health professionals work together to provide the best care possible to the patient. The UK Rare Diseases Framework has a rating of 2 in the EquiFrame framework because the policy not only states the Core Concept but also describes and explains how it can be achieved. For example, the policy demonstrates how implementation of virtual multidisciplinary team meetings, telemedicine and video appointments have supported better care coordination in the case of rare disease management (p. 14). Section title: Coordination of services Educational score: 1.5506956577301025 Domain: other Document type: Other Language: en Other policies from Australia, Hong Kong, UK, Finland and Canada each earned the rating of 1 as coordination of services and care is only stated or named in the policy as a strategy that sits under the overarching framework. For example, in the UK policy of Accelerating Genomic Policy in the NHS, the coordination of services and care is identified as an important task of NHS. The policy recommends the NHS achieve equity of access to genetic care by coordinating care, sharing of best practice and through driving a standardized model of delivery across the country (p. 45). Section title: Cultural responsiveness Educational score: 1.0730037689208984 Domain: other Document type: Other Language: en The Core Concept of cultural responsiveness is addressed in six reviewed policies (35%) from Australia and the UK. Each policy except WA Genomic Strategy earned the rating of 1 as they only mentioned the importance of the concept. The WA policy further provides details of what cultural responsiveness means and how it can be achieved. Apart from the WA policy, generally cultural responsiveness is a Core Concept that is not addressed in an in depth manner in the selected policies. Section title: Cultural responsiveness Educational score: 2.7170827388763428 Domain: biomedical Document type: Other Language: en While in the WA Genomic Health Strategy the term cultural responsiveness is not used, respecting ethnic, cultural and socio-economic diversity of patients and families is integral to its priority 1, achieving person and family centeredness (p.18). The policy highlights that respecting culture of Indigenous peoples is achieved by forming close partnerships with Indigenous community organizations (p. 21–22); the co-creation of genomic health services and policies therefore intrinsically recognizes the importance of community for Indigenous people and is a key aspect of cultural responsiveness. Importantly, the policy acknowledges that at present there is lack of trust within the Indigenous community towards genomic testing due to experiences of violations of their trust by early genomic initiatives. The policy therefore aims to develop standardized and culturally appropriate voluntary informed consent processes and uphold Aboriginal data sovereignty (p. 35). It also acknowledges the future need to explore the needs of consumers from various ethnic, social, and cultural groups (p. 25). Section title: Cultural responsiveness Educational score: 2.079408884048462 Domain: biomedical Document type: Other Language: en In the Australia National Health Genomics Policy Framework , cultural responsiveness is not addressed using the same terminology, however promoting awareness of genomics among culturally and linguistically diverse communities through culturally appropriate information is identified as a priority action. As barriers to equity are multifaceted, the policy recognizes that it is important to identify cultural acceptability barriers for genomics. Further, the policy also recognizes that evaluating the delivery of genomic health care in terms of its cultural appropriateness is an important priority action when delivering secure and responsive care to Aboriginal and Torres Strait Islander communities (p. 6). Section title: Cultural responsiveness Educational score: 1.501855731010437 Domain: biomedical Document type: Other Language: en The UK’s “Genome UK: The Future of Healthcare” policy identifies the importance of clinicians and researchers working together effectively to give individualized care for the patient which will include addressing cultural barriers (p. 26). Section title: Other core concepts that are addressed Educational score: 1.523795247077942 Domain: other Document type: Other Language: en The Core Concepts of capacity building and efficiency are addressed in six policies each (35%) from Australia, Canada, UK, Denmark, Hong Kong, India and Italy. The WA Genomics Strategy received a rating of 4 for the capacity-building Core Concept and a rating of 2 for efficiency, while Australia’s National Strategic Plan for Rare Diseases received a rating of 2 for capacity building in relation to ethnic minorities. All other policies include the two concepts as principles that direct the policy; they therefore did not receive ratings for these Core Concepts. Section title: Other core concepts that are addressed Educational score: 1.1202131509780884 Domain: other Document type: Other Language: en The Core Concepts of non-discrimination, individual services, privacy and prevention are addressed in five policies each (29%). They are addressed in policies from Australia, Canada, Denmark, Thailand and Italy. In each policy the Core Concept is stated but not explained in any detail and the policy did not detail how the Core Concept would be addressed. However, all five policies acknowledge the. Section title: Other core concepts that are addressed Educational score: 1.0613542795181274 Domain: other Document type: Other Language: en The Core Concepts of liberty and entitlement are not addressed in any of the 17 policies. Section title: Vulnerable communities Educational score: 1.337476134300232 Domain: other Document type: Other Language: en The coverage of vulnerable communities in the policies is sparse. Out of the 17 policies only eight (47%) specifically addressed vulnerable populations. In the other nine policies the Core Concept were addressed universally. Section title: Vulnerable communities Educational score: 1.6616264581680298 Domain: other Document type: Other Language: en All five Australian genomic policies included several vulnerable populations. Reference to Aboriginal and Torres Strait Islander communities were included in The National Strategic Action Plan for Rare Diseases ; National Health Genomics Policy Framework 2018–2021; Genetic and Genomic Healthcare for Victoria 2021: Improving the health and wellbeing of Victorians ; NSW Health Genomics Strategy: Implementation plan 2021–2025 ; and the WA Genomics Strategy 2022–2032: Towards precision medicine and precision public health . Three of the Australian policies (national plan for rare diseases, NSW genomic policy and WA genomic policy) stated specific policy actions that need to be taken to achieve better outcomes for vulnerable populations, and the WA policy also identified ways to monitor their progress. Section title: Vulnerable communities Educational score: 1.4741464853286743 Domain: other Document type: Other Language: en The Australian policies also identified culturally and linguistically diverse populations and communities who are living in remote and rural areas. For example, to achieve the Core Concept of individualized services with ethnic minorities the WA genomic policy also maps out strategies to monitor the progress of the Core Concept among the minority groups. In addition, the national action plan for rare diseases also includes people suffering from chronic diseases and people with limited resources. Section title: Vulnerable communities Educational score: 1.9311825037002563 Domain: biomedical Document type: Other Language: en The other three genomic policies that include vulnerable populations are from Canada and the UK. The Canadian Strategic Plan 2022–2027: Sequencing Our Future addresses five Core Concepts in relation to its Indigenous population, ethnic minority groups and people who live in remote areas. The policy not only states the vulnerable communities but describes in detail the context of these concepts in relation to the Vulnerable Groups. Genome UK: The Future of Healthcare includes ethnic minorities in addressing three Core Concepts. The policy focuses on the importance of obtaining participation of ethnic minority groups in genomic programs and identifies specific policy actions to address barriers for these groups. The UK rare disease framework also includes the above two vulnerable populations in relation to Core Concepts such as participation, coordination of services, cultural responsiveness and access. Section title: Recommendations Educational score: 3.564682722091675 Domain: biomedical Document type: Review Language: en This policy review highlights both the achievements and significant deficiencies in global genomic policies. It recognizes that genomic health science is rapidly evolving, presenting a major challenge for policies to remain current and effectively address new discoveries. Many of the policies that this desktop search captured from around the world focused on clinical genetic research. There is a relative dearth of policy that focused on clinical genetic services which may reflect a gap in policy and policy research translation and implementation. Further, it may reflect that the rapidity of genomic advances is accelerating past the traditional policy implementation and review processes and that there is a need for more culturally safe and responsive approaches. Section title: Recommendations Educational score: 3.24615216255188 Domain: biomedical Document type: Other Language: en Economic, socio-cultural and financial contexts do cause the recommendations for genomic policies to vary significantly among high income to middle income and low-income countries. Particularly in middle- and low-income countries there is a notable tension between striving for universal health equity and catering to specific local needs, often relegating genomic equity to a lower priority compared to high income countries. However, it is important to acknowledge that learning of genomic policy does not have to be a one-way stream from HIs to LMICs. Strength based approaches that are used for vulnerable populations such as Indigenous communities and solutions that are developed in LMICs are robust, practical, sustainable and efficient in low resource settings and can be quite effectively translatable in HIC settings ( 24–26 ). Section title: Inclusion of equity-focused core concepts in genomic policy Educational score: 4.037970542907715 Domain: biomedical Document type: Review Language: en The results of the desktop review of genomic policy demonstrate that the concept most frequently included in relation to achieving equity in genomic healthcare in policies is ‘access’. Previous literature points to a lack of clarity in the use of the concept of access in policy literature ( 38 ). Dimensions of access focused on in the policies vary according to the country demography and priorities. According to its social, economic and cultural contexts, the national genomic policies of Australia and the UK make reference to equity in access in terms of timeliness, location, economic and financial ability, availability of genomic services, age and ethnic and cultural background. The multifaceted nature of access that the Australian and English genomic policy describe aligns with the conceptualization of access by Levesque et al. ( 38 ) which contain five dimensions: Approachability; Acceptability; Availability and Accommodation; Affordability; and Appropriateness. Their conceptualization of access further aligns with how EquiFrame describes ‘access.’ Access in EquiFrame is defined in terms of the definition of access published by the United Nations Committee on Economic, Social and Cultural Rights which refers to the need for health facilities, goods and services without any discrimination. According to their definition, access further subdivides into physical, economic and information accessibility. Mannan et al. ( 19 ) state that policy has a main role to play in addressing issues with access to health care. Braveman and Guskin ( 15 ) argue that when assessing health equity, one needs to compare how health determinants affect more advantaged and less advantaged people differently and according to them this comparison becomes a reflection of how policies are on the course to achieve or not achieve equity in a population. Section title: Inclusion of equity-focused core concepts in genomic policy Educational score: 2.6621463298797607 Domain: biomedical Document type: Review Language: en In the Australian and UK genomic policies analyzed, access is addressed relative to the policies of other countries assessed in this review. They often reference the multifaceted nature of access: timeliness, location, economic and financial ability, availability of genomic services, age and ethnic and cultural background. However, in neither in the policies of Australia/UK nor of other countries, was access addressed beyond this explanation of its multiple dimensions. Specific policy actions to increase or achieve equity in access to genomic care and intensions or measurements to assess the intended improvement of access were missing in the policies. Section title: Barriers for participation Educational score: 1.225472331047058 Domain: other Document type: Other Language: en Participation, integration, quality, coordination of services and cultural responsiveness are the principles that are addressed to varying degrees in the policies that are reviewed. Section title: Barriers for participation Educational score: 3.6350057125091553 Domain: biomedical Document type: Review Language: en Participation is a concept that is commonly used in genomic policies particularly in high-income countries such as Australia, Canada and the UK. Participation is defined by WHO as the social involvement of the population in decisions that affect their health, defining their problem, health care services, implementation, evaluation and monitoring of those decisions ( 48 ). WHO contends that participation is a key driver of health equity as the employment of this principle facilitates the involvement of vulnerable communities as the agents and protagonists in the policies that directly affect them . In the reviewed policies the terms engagement, patient voice and lived experience of patients are used interchangeably with participation. In the genomic policies where this Core Concept is addressed, there are two distinct areas that the focus is on in relation to obtaining participation of the communities: genomic research and policy implementation. Section title: Barriers for participation Educational score: 3.404130458831787 Domain: biomedical Document type: Study Language: en A common theme observable in the genomic policies of the UK and Australia (WA) is acknowledgement of the historical reasons for the reluctance to engage with genomic health system among vulnerable populations. This is compounded by assumptions that health professionals have about minority communities. For example, previous literature demonstrates that health professionals make assumptions about socio-cultural, economic and biological characteristics of ethnic and racially different minorities that effects their care ( 28 ). This two-way barrier has led to a lack of diversity in available datasets across high income countries, which in turn impacts negatively on the ability of minority populations to obtain benefits of genomic medicine and healthcare ( 2 ). Section title: Barriers for participation Educational score: 2.052385091781616 Domain: biomedical Document type: Other Language: en To increase participation in genomic research or care from ethnic and other minority groups, genomic policies need to recognize and address this two-pronged barrier effectively. The ‘Accelerating Genomic Medicine in the NHS’ ( 43 ) policy sets forth a model to follow to achieve this outcome. In the reviewed genomic policies, there is an absence of recognition and weight given to the capacity that participation has, to function as a driver for equity. Section title: Barriers for participation Educational score: 2.4032084941864014 Domain: biomedical Document type: Other Language: en Participation is mentioned in the reviewed policies mainly in relation to research and policy implementation. However, participation as a strategy to achieve equity in genomic health care needs to be included from the first step itself of policy development including in research and policy implementation. This brings our attention to an important measure that needs to be taken in future policy making in genomic health care: the need to include participation of vulnerable communities and populations in all stages of policy process that includes the stages of problem diagnosis, planning, implementation, evaluation and monitoring. Health inequity and inefficiency are significant negative consequences that irrefutably follow, in the instance when this is not done ( 48 ). Section title: Integration as a strategy for equity Educational score: 3.203205108642578 Domain: biomedical Document type: Other Language: en Integration of genomic healthcare in the mainstream health system is the most notable strategy and service model that many policies recommend in relation to the Core Concept of integration. Integration of health services is recognized as a mechanism that facilitates and enhances health equity ( 49 ). WHO contends that integration as a principle encourages the prioritization and selection of health services to deliver a holistic care over the life course of a population that include processes related to prevention, protection, promotion, diagnosis, treatment and management of diseases . Section title: Integration as a strategy for equity Educational score: 3.387651205062866 Domain: biomedical Document type: Other Language: en Regarding the principle of integration, Australian and English genomic policy in particular read strongly according to the EquiFrame framework: they all focus on giving clearly articulated, specific policy actions to integrate genomic healthcare into the mainstream health system. Within the principle of integration, Australian policies identify focus areas that need to be included in it such as the inclusion of education, workforce knowledge about genomics, data security and sharing, high quality and safe services and person-centered approach as strategies to employ. These aspects of integration are also endorsed by WHO ( 49 ). The examination of integration in Australian genomic policy provides a relatively complete model of the ways and steps that can be taken to achieve genomic care equity through integration. While the integration of the above sectors is important in genomic health care, it is also important to acknowledge the need for integration of the overarching processes of prevention, protection, and promotion. The developments of genomic medicine and healthcare have a significant impact on these processes. Section title: Quality of care and equity Educational score: 3.9494378566741943 Domain: biomedical Document type: Review Language: en Quality of care is integral to health equity. In fact, effectiveness, people centeredness, equity, timeliness, safety, integration and efficiency are widely recognized to be the characteristics of quality of care ( 29 ). There is a growing body of literature that argue that quality of care is achieved only if it is examined through an equity lens ( 30 ). Equity and quality of care in health services should not depend on age, sex, gender, race, indigeneity and ethnicity, migrant status, geographical location, socio economic status, language, religion and disability and other socio-economic and cultural factors ( 30 , 31 ). The delivery of equitable and high-quality health care demands an understanding of the complex interplay of the above factors ( 30 ). The reviewed policies recognize quality of care as an outcome of strong senior leadership in the health system and an outcome of person-centered genomic healthcare, improved safety, cost effectiveness and of a skilled and knowledgeable health workforce. However, in these policies, there is little acknowledgment given to the understanding of the interplay of various socio-economic, cultural and geographic aspects in relation to achieving quality of care in genomic healthcare. Section title: Cultural responsiveness as an afterthought Educational score: 3.948762893676758 Domain: biomedical Document type: Review Language: en Cultural responsiveness is one of the Core Concepts that is sparsely addressed in the reviewed policies. Naturally, cultural responsiveness becomes an important necessity in health policy where there is a significant culturally diverse population; this Core Concept is seen in most Australian and English genomic policies. Forming close relationships with the communities of Indigenous and ethnic minorities, developing culturally appropriate voluntary informed consent processes, increasing cultural awareness in genomic clinicians and researchers are some of the policy actions that can be seen in them to increase cultural responsiveness. Recognition of culture is a cornerstone of strength-based approaches used to achieve health equity in marginalized and vulnerable communities ( 27 ). Cultural responsiveness is an important Core Concept that needs to be included in genomic policies as literature has already demonstrated that the assumptions that health professionals tend to make of patients from minority communities function as a significant barrier for accessing genomic healthcare ( 28 ). One’s culture and language act as major factors that impact on the quality of the healthcare a patient receives, health outcomes and patient satisfaction ( 36 ). Although cultural diversity and cultural competence are recognized as important obligations that the health services need to meet to address the needs of ethnic minorities and Indigenous communities, literature identify systemic, organizational, professional and individual challenges that health services encounter when delivering culturally responsive care ( 37 ). Genomic policies that were reviewed give little focus on the importance to the cultural responsiveness when addressing the needs of ethnic minority and Indigenous communities. Section title: Cultural responsiveness as an afterthought Educational score: 1.5067782402038574 Domain: other Document type: Other Language: en Other Core Concepts such as prevention, liberty and entitlement were either addressed in very few policies or were not addressed at all. However, all three concepts are intrinsically linked to the provision of genomic care and to factors such as confidentiality, privacy and data sovereignty. Section title: Missing CCs and implications Educational score: 1.4704201221466064 Domain: other Document type: Other Language: en Liberty and entitlement are not included in any of the selected policies. However, the gravity and the negative consequences of historical exclusion of the CC of liberty, especially from genomic policies related to vulnerable groups such as Indigenous populations and ethnic minorities, are clearly documented in the selected policies. Liberty for example is defined by Turnbull and Stowe ( 32 ) as a constitutional principle that gives the right for persons for certain entitlements and freedoms that include physical and general freedom to conduct one’s life as one prefers to. Considering the historical damaging of trust that has taken place due to unwarranted and unconsented genetic initiatives with Indigenous people in Australia, it would be detrimental and harmful for not including the CC of liberty in future genomic policy. Section title: Missing CCs and implications Educational score: 2.6567957401275635 Domain: biomedical Document type: Other Language: en According to Turnbull and Stowe ( 32 ) entitlement is one’s right to receive services according to one’s strengths and resources, to be served for one’s benefit to the utmost extent possible. Entitlement is a CC that could be considered as indispensable for genomic policies, especially at present time where irrespective of vulnerability, awareness of genomic services is considered low in the general populations. According to Goddard and Smith ( 33 ) the CC of entitlement would not be present in a situation where there is a lack of awareness and clarity of the availability of a specific health service by all population groups. Therefore, entitlement is a CC that future policy reviews of genomics will have to focus on especially in contexts where there is unequal awareness and knowledge of genomics services in the population. Section title: Vulnerable communities Educational score: 1.199981689453125 Domain: other Document type: Other Language: en Only about a quarter of the reviewed policies included vulnerable communities; most policies mentioned the Core Concepts universally. The most noted vulnerable communities in the policies are Indigenous and CALD communities and communities that live in rural and remote areas. Although in many contexts these populations also overlap with economically and financially deprived groups, the exclusion of the latter in policy could result in such vulnerable groups being overlooked by services. Section title: Vulnerable communities Educational score: 1.912817120552063 Domain: other Document type: Other Language: en Recent literature on vulnerability and vulnerable groups emphasizes the importance of promoting an intersectionality framework to use as a guiding principle to study vulnerable individuals and groups because vulnerability is a nuanced concept and cannot be rigidly applied to all in a socio-demographic group ( 50 ). However, in EquiFrame framework this nuanced nature of vulnerability of groups of people is not captured. Section title: Vulnerable communities Educational score: 2.890913248062134 Domain: biomedical Document type: Review Language: en This policy review focuses on the level of inclusion of equity in genomic policies mainly in high income countries such as Australia, England, Canada, Hong Kong, Finland and Denmark. Genomic policies are not common in middle and low income and developing countries. This demonstrates that genomic health is still an area in healthcare that is limited to the high-income countries and there is a long and windy road ahead of low- and middle-income countries to include the benefits of discoveries made in genomic care in health policy. Section title: Vulnerable communities Educational score: 2.135449171066284 Domain: biomedical Document type: Other Language: en However, it is important to acknowledge that there is a significant body of literature that point out to the effectiveness and translatability of many low cost and high impact innovations that are employed in LMICs especially to tackle their high health care costs which could be adopted to address problems such as inequity in health in HICs ( 24–26 , 34 ). Section title: Limitations Educational score: 2.727743625640869 Domain: biomedical Document type: Study Language: en Although EquiFrame is a practical tool that policymakers and reviewers can use to assess a policy, it also presents few limitations. Even though the framework was developed to review policy in low- and middle-income countries, the policies reviewed in this article come mostly from developed, high income countries. Therefore, in relation to the Core Concepts, there could be others we may have missed that are only captured in policy from developed countries. The other limitation is the inclusion of vulnerable populations. The identification and determination of vulnerable population groups could vary widely in different contexts ( 35 ). For example, Ivanova et al. ( 22 ) argue that identification of a vulnerable group is a complex process as there is no single criteria or indicator to benchmark or measure vulnerability of a group of population ( 22 ). Genomics is a highly specialized field of health service. Colonial histories and related issues such as racism and discrimination render certain groups such as Indigenous populations particularly vulnerable and could make them mistrust services that offer genetic services ( 51 ). Therefore, to capture these populations, we have included Indigenous population group in addition to the given vulnerable groups to make the list more inclusive. Section title: Limitations Educational score: 1.3846734762191772 Domain: other Document type: Review Language: en Further, even though law, legislation and acts can have a direct impact on achieving equity in health services, we have excluded them from this review, due to their varied and multitude nature across countries and the lack of easy accessibility. We sought to contain this analysis only to policies of genomic services. Therefore the exclusion of laws, legislations and acts from this review can also be considered as a limitation. Section title: Conclusion Educational score: 3.6538333892822266 Domain: biomedical Document type: Review Language: en This policy review reveals that with few exceptions, genomic policies across high or middle-income countries fall significantly short in outlining specific, actionable steps toward achieving equity. Given that genomics is an evolving field with rapid advancements in discoveries and clinical applications, it is understandably challenging for policies to stay abreast of these developments. Ensuring that the benefits of these advancements are equitably distributed poses a further challenge. Therefore, policymakers are tasked with the dual responsibility of not only keeping policies current with the fast-paced evolution of genomics but also ensuring these advances are accessible and beneficial to all, thereby maintaining a balance between innovation and equity in genomic healthcare. | Review | biomedical | en | 0.999996 |
PMC11695416 | Section title: Introduction Educational score: 4.221561431884766 Domain: biomedical Document type: Study Language: en The Japanese encephalitis virus (JEV) is a notable member of the mosquito-borne viruses in the Flaviviridae family, responsible for the most prevalent form of encephalitis in humans and horses across the Asia-Pacific region ( 1 ). This region includes approximately 24 countries, including the two most populous nations, China and India, as well as several densely populated countries such as Singapore and Bangladesh ( 2 , 3 ). JEV is primarily transmitted by mosquitoes of the genus Culex ( 4 ). Pigs and wading birds play key roles in maintaining viral circulation and transmitting the virus to humans and horses ( 6 , 7 ). Annually, an estimated 68,000 to 100,000 clinical cases of Japanese encephalitis (JE) occur worldwide, resulting in approximately 15,000 to 25,000 fatalities ( 8 , 9 ). JEV is emerging in new areas, with reported cases in Europe and Africa ( 10 , 11 ). Section title: Introduction Educational score: 3.986358404159546 Domain: biomedical Document type: Review Language: en Distinguishing JE from other encephalitis cases is challenging, making laboratory confirmation essential ( 12 ). The WHO recommends diagnosing JE using the IgM-capture ELISA (MAC-ELISA) from a single cerebrospinal fluid (CSF) or serum sample, with CSF preferred to reduce false positives from prior infections or vaccinations ( 13 ). Other tests, such as the hemagglutination inhibition (HI) test, indirect immunofluorescence assay (IFA), plaque reduction neutralization test (PRNT), and nucleic acid detection, may also be used ( 14 , 15 ). There is no specific antiviral treatment for JEV; management is supportive, focusing on nutrition, airway maintenance, and anticonvulsants for seizure control ( 16 – 18 ). Vaccination can effectively prevent JEV infection in both humans and animals ( 19 , 20 ). Section title: Introduction Educational score: 4.042291164398193 Domain: biomedical Document type: Study Language: en Live attenuated and inactivated vaccines against JE have been available for decades ( Table 1 ), yet JEV remains the leading cause of viral encephalitis in Asia ( 23 ). Approximately 3 billion people in endemic regions are at risk, but only 80–100 million JE vaccine doses are produced annually, highlighting a significant gap in coverage ( 21 ). Vaccination costs range from $1.15 to $2.41 per child per dose ( 24 – 26 ). A study in the Philippines found that managing JE illness can cost between $859 and $1,209 per case. JE’s impact drives vaccine demand, increases healthcare costs, and emphasizes the need for public health initiatives and further vaccine development. Its spread into new territories also raises global health security concerns, prompting greater international collaboration and funding for surveillance and response ( 24 , 27 ). Section title: Introduction Educational score: 4.077493667602539 Domain: biomedical Document type: Study Language: en Live-attenuated vaccines induce strong immune responses but can be risky for immunocompromised individuals ( 28 ), while whole inactivated vaccines are safer but often have short-lived efficacy ( 29 ). Both require large-scale pathogen cultivation, leading to high costs and biosafety concerns ( 28 , 30 ). Furthermore, traditional JEV vaccines offer limited cross-protection against different strains due to the virus’s genetic variability, undermining their efficacy against emerging variants ( 15 , 30 ). Consequently, there is an urgent need to develop a safe, stable, durable, and cost-effective vaccine that elicits robust immunogenicity across diverse JEV strains. Recombinant pathogen-derived vaccines, coupled with nanoparticle (NP) delivery systems, present a promising solution to meet this critical need ( 31 ). Section title: Introduction Educational score: 4.071580410003662 Domain: biomedical Document type: Review Language: en Nanoparticle vaccines represent a breakthrough in vaccinology, offering enhanced immune responses, safety, stability, and adaptability. While research is ongoing, these vaccines show significant promise in addressing a wide range of infectious diseases more efficiently than traditional vaccines. NPs inherently contain pathogen-associated molecular patterns (PAMPs) that are recognized by the immune system as signals of potential danger, thereby improving antigen presentation and enhancing immune responses ( 32 ). Additionally, nanoparticles can carry multiple antigens, enabling the development of multivalent vaccines. Types of NPs, such as protein nanocages, outer membrane vesicles (OMVs), virus-like particles (VLPs), and polymeric NPs, effectively overcome barriers to recombinant vaccine delivery ( 31 ). Section title: Introduction Educational score: 4.553882598876953 Domain: biomedical Document type: Study Language: en During NP vaccination, innate immune cells, such as antigen-presenting cells (APCs) like dendritic cells (DCs) and macrophages, detect and engulf NPs, triggering antigen presentation on MHC Class I and II molecules and activating T-helper (CD4+) and cytotoxic (CD8+) T cells ( 31 – 35 ). CD4+ T cells promote B-cell differentiation and antibody production, which inhibit viral replication ( 36 ), while CD8+ T cells eliminate infected cells when humoral immunity is insufficient ( 36 – 38 ). After endosomal uptake, NPs release antigens into the cytoplasm, activating CD8+ T cells to target and destroy infected cells ( 39 ). NP delivery systems also stimulate T- and B-cell memory, contributing to a long-lasting immune response ( 40 ). Recombinant vaccines are particularly advantageous for incorporating a limited set of B- and T-cell epitopes, as their ability to precisely engineer specific immunogenic components results in targeted, efficient immune activation ( 41 ). By focusing on well-characterized epitopes conserved across JEV strains, developers can elicit stronger immune responses, reduce immune tolerance, and potentially achieve cross-protection against diverse strains ( 31 ). Encapsulating these epitopes in NPs further enhances immune responses, minimizes adverse effects, and enables cost-effective production ( 42 ). Section title: Introduction Educational score: 3.872631788253784 Domain: biomedical Document type: Review Language: en Nanoparticle vaccine technology has progressed from laboratory research to clinical application. Outer membrane vesicles (OMVs) and virus-like particles (VLPs) have received approval from the Food and Drug Administration (FDA) and are currently available on the market, while additional vaccines are under investigation for safety and efficacy ( 43 ). Researchers are developing a nanoparticle vaccine for JEV, with promising results, though it has not yet reached clinical trials or FDA approval. This review consolidates findings on nanoparticle vaccine development for JEV, with the aim of enhancing understanding and guiding future research. Section title: Literature search and study selection Educational score: 4.140002250671387 Domain: biomedical Document type: Review Language: en The methodology outlined in the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) declaration was followed in the design and preparation of this systematic review ( 44 ). English-language databases, including PubMed, Web of Science, ScienceDirect, Google Scholar, Scopus, and EBSCO , were systematically explored for research articles published between 2000 and 2023 that focused on the development of candidate nanoparticle vaccines against JEV with in vivo /animal model immunogenicity evaluation. Keywords such as “Japanese encephalitis,” “nanoparticle,” “virus-like particle,” “self-assembled particle,” “immunogenicity,” “antigenicity,” “vaccine,” and “candidate vaccine” were included in the comprehensive search. Additional searches were conducted by reviewing the bibliographies of relevant primary and review publications. Furthermore, a thorough manual search of the papers’ full reference lists was carried out to ensure no articles were overlooked. Full-text articles published in the aforementioned databases and those evaluating the proposed vaccine in vivo /animal models were included. We excluded gray literature and abstracts of papers presented at conferences. After a thorough evaluation of the articles, studies were excluded if they met any of the following criteria: (1) studies that did not focus on vaccine candidates that form nanoparticles; (2) studies that were not available in full text; (3) studies not published in English; (4) reviews or descriptive studies; and (5) articles that lacked sufficient information about the vaccine’s immunogenicity and level of protection in an animal model. Section title: Data extraction Educational score: 3.9598257541656494 Domain: biomedical Document type: Study Language: en The types of nanoparticles (NPs), antigenic components, expression systems, virus strains used for challenges, NP sizes, doses for single immunization, adjuvants, number of booster doses, animal models, routes of administration, positive control vaccines, methods for assessing immunogenicity, and key results regarding immunogenicity were systematically compiled in a well-structured data extraction Excel sheet. Additionally, bibliographic details, including authors and publication years, were documented. Section title: Analysis of the included literature Educational score: 4.013772010803223 Domain: biomedical Document type: Review Language: en This systematic review included a total of 28 articles published from 2001 to 2023 that studied potential candidate vaccine development against JEV by incorporating particulate antigens into NPs. Different NP formation techniques were employed in these studies : 16 VLPs, 3 Sub viral particles (SVPs), 2 chitosan-based NPs, and other NPs, including one study each on colloidal gold, bio-nanocapsules, lumazine synthase-assembled particles, lipid-based nanoparticles (LNPs), AB5-type toxin-based nanoscaffolds, γ-PGA, and polyethylene glycol-precipitated NPs. Section title: Size and antigenic components of NPs Educational score: 4.038336753845215 Domain: biomedical Document type: Study Language: en The primary goal of developing nanoparticle-based vaccines is to deliver antigenic components that structurally and functionally resemble live pathogens. These antigens cannot replicate or cause infection, but they contain immunogenic elements of the pathogen that can be recognized by APCs and are essential for fully activating the immune system ( 43 ). Therefore, it is commonly understood that particles containing antigenic components and sized similarly to viruses are recognized by the body as viruses. Seven of the 28 compiled articles did not specify the size of the developed NPs. The size of the NPs varied significantly across different antigen-particulate approaches used in the articles. For example, chitosan-based NPs ranged from 200–333 nm, while VLPs and sub-viral particles ranged from 18–200 nm and 25–30 nm, respectively. The remaining NP types, which ranged in size from 30 to 80 nm, were used in only one study. To better understand the variations in NP size, the error bar provides an overview of how nanoparticle sizes differ across various antigen-particulate techniques. Section title: Size and antigenic components of NPs Educational score: 4.013246536254883 Domain: biomedical Document type: Study Language: en Nanoparticles serve as carriers to facilitate the attachment of antigens to their surface, thereby enhancing immune response by promoting efficient trafficking and recognition by cellular receptors ( 73 ). Structural proteins, mRNA or DNA encoding these proteins, as well as inactivated or live whole pathogens, were utilized as antigenic components in the development of NP-based vaccines in the studies reviewed . Among the 28 publications examined, Pre-membrane and Envelope (prM and E) combined protein, E protein, and E domain-III protein were employed in 12 (42.86%), 7 (25%), and 4 (14.3%) studies, respectively. Additionally, single articles featured the use of Capsid, Pre-membrane and Envelope (CprME) proteins, mRNA of prM & E proteins, DNA of prM & E proteins, attenuated JEV, and inactivated JEV as antigenic components. Section title: Expression systems of the recombinant protein Educational score: 3.949725866317749 Domain: biomedical Document type: Review Language: en The expression of recombinant proteins is a pivotal element in the development of antigenic protein-based NP vaccines. Over the years, recombinant protein expression has been successfully carried out in a variety of host systems, including yeasts, bacteria, plants, transgenic animals, as well as cultured insect and mammalian cells ( 74 ). In the studies reviewed, E. coli , HEK-293T, BHK-21, and RK-13 cells were employed to express recombinant JEV antigenic proteins, with respective study frequencies of 7, 4, 4, and 3. Additional expression systems used in individual studies included yeast ( Pichia pastoris strain X33), silkworm Bm-N cells, COS-1, HS-deficient CHO cells, C6/36, and AP-61 cells, as well as silkworm pupae . Section title: Laboratory animals Educational score: 3.974281072616577 Domain: biomedical Document type: Review Language: en Mice are the most commonly used animals in JEV vaccine development and pathogenesis studies, despite variations in their susceptibility depending on factors such as viral strain, inoculum dose, route of administration, and age ( 75 , 76 ). All studies included in this review used mice as the animal model; however, swine and rabbits were also utilized alongside mice in 3 and 2 studies, respectively. A total of six specified mouse strains, as well as one unspecified strain, were employed across the studies (see Table 2 ). BALB/c mice were used in the majority of studies ( 19 ), while C57BL/6 and ICR strains were each used in 3 studies, and C3H/He, ddY, and FVB/J strains were used in 2, 1, and 1 study, respectively. Section title: Dosage and routes of administration Educational score: 4.050199508666992 Domain: biomedical Document type: Review Language: en For new vaccine candidates, dose-response studies are often conducted to determine the optimal dose for inducing the highest antibody production in animal models. Several studies ( 45 , 47 , 50 , 61 , 67 , 68 ) included in this review carried out dose-optimization experiments at the animal model level during the development of NP-based JEV vaccines. Another key aspect of dosage evaluation is determining whether a single immunization or additional booster doses, along with adjuvants, are necessary to elicit a protective immune response. In nanoparticle-based vaccines, synthetic nanoparticles, such as inorganic and liposome-based particles, typically require multiple doses and the addition of adjuvants, while biologically derived nanoparticles often have intrinsic properties that enhance their ability to stimulate the immune system independently, reducing the need for artificial adjuvants ( 43 ). Consequently, many of the studies reviewed focused on self-assembled VLPs and sub-viral particles, which required booster immunizations and the use of adjuvants. Of the 28 articles reviewed, 4 studies used only a single-dose immunization, while 7 studies did not incorporate adjuvants. The remaining studies administered 1–3 booster doses and utilized various adjuvants, including Alum, Freund’s, Montanide ISA50V2 & IMS-1313, and Cytosine-phosphoguanine (CpG). Section title: Dosage and routes of administration Educational score: 3.9650766849517822 Domain: biomedical Document type: Review Language: en The adjuvanticity of nano-vaccines, which is influenced by the route of administration, depends on the specific properties of the antigen-carrying nanoparticles. For example, the slow degradation of hard nanoparticles promotes antigen uptake when administered intravenously (I.V.), while soft nanoparticles are more efficient at stimulating antigen uptake when delivered via subcutaneous (S.C.) injection ( 76 ). However, testing of new vaccines typically begins in small animal models, which have different anatomies from humans or other animals, and as a result, the route of administration cannot be fully evaluated in these models. Thus, studies using different vaccine administration routes in small animals are insufficient for determining the most effective immunization routes for other animals or humans ( 77 ). Researchers in the studies reviewed have used various routes of administration for their candidate NP vaccines, including intramuscular (I.M.), subcutaneous (S.C.), intradermal (I.D.), intraperitoneal (I.P.), and intranasal (I.N.) ( Table 2 ). Section title: Immunogenicity assessment methods Educational score: 4.299027919769287 Domain: biomedical Document type: Review Language: en To obtain “proof of concept” data supporting clinical trial development, assessing the immunogenicity of potential vaccines in animal models is essential ( 78 ). The induction of adaptive immunity can be evaluated through humoral or cellular immune responses. The most widely used methods for evaluating vaccine immunogenicity include seroconversion rates, neutralizing antibody titers, and immune cell proliferation assays ( 79 ). Additionally, the efficacy of new vaccines is typically evaluated based on protection against a lethal dose of the pathogen. As indicated in Table 2 , the majority of the reviewed studies assessed immunogenicity using virus-neutralizing assays, immunoglobulin G (IgG) titers, and protection against lethal dose challenges. Virus-neutralizing and seroconversion assays are crucial for vaccine evaluation, as they quantify neutralizing antibodies that inhibit viral infections and indicate potential protective immunity ( 2 ). However, these assays primarily focus on antibody responses, potentially overlooking other key components of immunity, such as T-cell responses and non-neutralizing antibodies, which also contribute to protection ( 80 , 81 ). Non-neutralizing antibodies enhance immunity by facilitating opsonization, activating the complement system, and promoting antibody-dependent cellular cytotoxicity (ADCC), which targets infected cells for destruction. Additionally, non-neutralizing antibodies influence immune cell activation and differentiation, shaping the overall immune response. While not directly neutralizing pathogens, these antibodies play critical roles in pathogen clearance and immune regulation ( 82 ). Section title: Immunogenicity assessment methods Educational score: 4.295670986175537 Domain: biomedical Document type: Study Language: en Most of the recent studies also included lymphocyte proliferation assays ( 62 , 63 , 65 , 68 , 70 , 71 ) and cytokine assays to further evaluate vaccine immunogenicity ( 52 , 61 , 63 , 70 , 83 ). Lymphocyte proliferation assays are essential for assessing immune responses to new vaccine candidates, as they measure the activation and expansion of T and B cells upon antigen exposure. Using techniques like radiolabeled thymidine incorporation or flow cytometry, these assays evaluate T-cell activation and differentiation into effector and memory cells ( 84 ). A robust proliferation response indicates effective immune activation and suggests strong immunogenicity ( 85 , 86 ). However, these assays do not directly assess the functionality or effectiveness of the immune response, such as antibody or cytokine production. Therefore, while lymphocyte proliferation assays provide valuable information, they should be complemented with other immunological assessments for a comprehensive evaluation of vaccine efficacy ( 87 ). Cytokine assays are also crucial for evaluating immunogenicity, as they measure key cytokines that reflect immune cell activation. Cytokines like IL-12, TNF-α, IFN-γ, and IL-4 help assess the strength and type of immune response, including Th1 (cellular immunity) and Th2 (humoral immunity) responses, which are critical for vaccine efficacy ( 88 , 89 ). Cytokine assays also provide insights into immune memory by detecting markers such as IL-2 and IFN-γ, which are associated with memory T-cell differentiation ( 90 ). Section title: Immune response and protection Educational score: 4.274669170379639 Domain: biomedical Document type: Review Language: en This systematic review reveals that all of the studies reviewed employed a virus-neutralizing assay, and in nearly all cases, the candidate vaccines elicited higher neutralizing antibody titers. Additionally, elevated levels of IgG, IgG1, and IgG2a were observed in immunized mice ( 50 , 54 , 60 , 63 , 68 ) ( Table 2 ). The VLP-based candidate vaccine, engineered from JEV prM and E proteins in BHK-21 cells, induced higher neutralizing antibody titers compared to live attenuated vaccines ( 45 ). VLPs mimic the viral structure, enhancing antigen presentation and boosting immune responses ( 91 ). They also stimulate humoral immunity through a T-helper cell-independent pathway ( 92 ) and activate CD8+ cytotoxic T cells via the MHC class I pathway, bypassing the need for extracellular antigens ( 93 ). This process involves antigen uptake by CD8− dendritic cells and transfer to secondary lymphoid organs, where it is presented via TAP-dependent and independent pathways ( 94 ). Similarly, γ-PGA-based nanoparticles containing inactivated JEV ( 55 ) induced higher neutralizing antibody titers than live vaccines. γ-PGA stimulates innate immunity, promotes Th1 responses, and enhances cytotoxic T lymphocyte (CTL) activity ( 95 ). Its adjuvant properties further amplify the immune response by activating antigen-presenting cells and T cells, leading to increased antibody production and stronger immune memory ( 96 ). Section title: Immune response and protection Educational score: 4.447524070739746 Domain: biomedical Document type: Study Language: en Cytokine concentrations induced by the NP vaccines were also analyzed, with results showing elevated levels of IL-4, IL-12, IFN-α, and IFN-γ ( 61 , 63 , 70 , 83 ). The candidate NP vaccines effectively activated both innate immune cells (dendritic cells and macrophages) and adaptive immune responses, providing robust protection against pathogens. Activation of APCs stimulates pro-inflammatory cytokine production, primarily through PRRs such as TLRs ( 97 , 98 ). This activation drives the differentiation of CD4+ T cells into Th1 and Th2 subsets: IL-12 from APCs promotes Th1 differentiation and IFN-γ production, while IL-4 drives Th2 differentiation, enhancing antibody responses ( 32 , 99 , 100 ). NPs also stimulate type I interferon production, including IFN-α, through signaling pathways activated by viral components or adjuvants. Cytokines such as IL-12 and IL-4 create feedback loops that amplify cytokine production, with IFN-γ further stimulating IL-12 production. Enhanced antigen presentation by NPs improves T-cell activation and cytokine production, particularly for CD8+ cytotoxic and CD4+ helper T cells ( 101 ). This cross-talk between immune cells leads to a coordinated cytokine response, contributing to improved vaccine efficacy and protection ( 102 , 103 ). Section title: Immune response and protection Educational score: 4.317696571350098 Domain: biomedical Document type: Study Language: en Furthermore, the candidate JE NP vaccines induced significant T-cell proliferation in the spleen ( 62 , 65 , 71 ). This immune cell proliferation is linked to the process in which, upon antigen administration, APCs present processed antigen peptides on MHC molecules, which is critical for T-cell activation. Naive T cells in lymphoid tissues encounter these antigen-MHC complexes, triggering the first signal necessary for activation. For full activation, T cells also require co-stimulatory signals from APCs, such as the interaction between CD80/CD86 on APCs and CD28 on T cells ( 104 ). Additionally, activated APCs secrete inflammatory cytokines, including IL-12, IL-6, and TNF-α, which further enhance T-cell activation and proliferation. Other cytokines, such as IFN-γ and IL-4, promote T-cell differentiation into effector subsets, increasing their proliferative capacity and the number of antigen-specific T cells in lymphoid tissues. Some of these proliferating T cells differentiate into memory T cells, which persist and provide long-term immunity, ensuring a rapid response upon re-exposure to the antigen ( 37 ). These findings suggest that NP-based candidate vaccines can effectively enhance both humoral and cellular adaptive immune responses against JEV. The next-generation NP vaccines provided 70–100% protection for immunized mice against a lethal dose of virulent JEV, despite variations in adjuvants, doses, NP types, antigens, and animal models used across studies ( Table 2 ). Section title: Discussion Educational score: 4.381836414337158 Domain: biomedical Document type: Study Language: en Developing vaccines against viral infections is generally more straightforward than creating antiviral drugs, and vaccines are far more effective at preventing disease progression before significant damage occurs. JE is viral encephalitis for which no proven antiviral treatment exists; therefore, vaccination remains the primary strategy for effectively controlling and preventing the disease. As noted in the introduction, both live-attenuated and inactivated vaccines are utilized in several countries and play a crucial role in JE prevention ( 20 ), despite concerns regarding cost, safety, and challenges related to cross-protection ( 30 ). The high genetic and antigenic variability of the JEV complicates the development of a universally protective vaccine. JEV’s diverse genotypes, which exhibit variations in the E protein, result in differences in immune recognition, making vaccines designed for specific strains less effective against others ( 15 , 105 ). For instance, while inactivated and live-attenuated vaccines show strong efficacy against genotype III, they offer reduced protection against genotype I ( 106 , 107 ). This variability, along with the risk of immune escape due to antigenic mutations, presents significant challenges in developing a universal JEV vaccine ( 108 ). Section title: Discussion Educational score: 4.194740295410156 Domain: biomedical Document type: Review Language: en To address these issues, recombinant vaccines present a promising solution, as they enable precise targeting of conserved epitopes across multiple JEV strains. Using recombinant DNA technology, specific viral proteins, such as the E protein from different strains, can be produced and purified. This reduces the risk of immune escape and facilitates the creation of multivalent vaccines that provide broader protection ( 31 , 41 ). The adjuvant requirements and early degradation problems often associated with recombinant vaccines can be mitigated through nanoparticle-based delivery systems ( 39 , 109 ). By encapsulating multiple epitopes from different JEV strains, nanoparticles can contribute to the development of multivalent vaccines that offer more durable and comprehensive immunity, effectively addressing cross-strain protection. Thus, the combination of recombinant vaccine technology and nanoparticle delivery systems holds considerable promise for overcoming JEV’s genetic diversity and enhancing global vaccine coverage ( 31 ). This review consolidates findings on the development of nanoparticle-based vaccines for JE, aiming to improve understanding and guide the future development of safe, effective, multivalent, and affordable vaccines. A variety of nanoparticles, including self-assembled proteins, biological polymers, synthetic compounds, and inorganic nanoparticles, are being explored as antigen carriers for JE vaccines ( 110 , 111 ). Section title: Self-assembled protein-based JE NP vaccines Educational score: 4.065032482147217 Domain: biomedical Document type: Review Language: en Over 50% of the reviewed articles focused on VLP-based vaccine development, utilizing structural JEV proteins as antigens. Figure 5 provides an overview of the antigenic components, adjuvants, animal models, and administration routes used in JE VLP vaccine studies. Recombinant technology enables the in vitro production of viral structural proteins ( 112 ), leading to the formation of smaller entities known as sub-viral particles, which elicit robust innate, humoral, and cellular immune responses in both animals and humans ( 113 ). The majority of these in vitro -generated sub-viral particles retain the characteristics of VLPs, composed of one or more full-length viral structural proteins. In contrast, some consist of smaller sub-viral particles formed from truncated capsid proteins ( 114 ). Section title: Self-assembled protein-based JE NP vaccines Educational score: 4.279757499694824 Domain: biomedical Document type: Study Language: en In conjunction with mRNA and viral vector-based vaccines, VLP technology provides an alternative platform for developing effective vaccines against major infectious diseases. Both SVPs and VLPs elicit robust immune responses by activating innate and adaptive immunity. Upon uptake by DCs and other APCs, VLPs and SVPs trigger TLR-mediated signaling pathways, promoting the release of pro-inflammatory cytokines and enhancing antigen presentation ( 115 ). Their repetitive structure efficiently activates B cells, leading to the production of high-affinity antibodies that neutralize pathogens ( 116 ). Additionally, these particles stimulate CD4+ T-helper cells, which facilitate B cell differentiation and activate CD8+ cytotoxic T cells via cross-presentation, offering strong protection against infections ( 117 ). Moreover, the particulate nature of VLPs and SVPs enhances long-term immunity by promoting the formation of memory B cells and T cells ( 118 ). Section title: Self-assembled protein-based JE NP vaccines Educational score: 3.9878833293914795 Domain: biomedical Document type: Review Language: en Virus-like particles (VLPs) are considerably more immunogenic due to their repetitive antigenic epitopes, which provide a more authentic signal for immune system recognition. As summarized in the main findings section ( Table 2 ), VLP-based candidate vaccines, both with and without adjuvants, were able to elicit immune responses comparable to those induced by live and inactivated vaccines. Five studies—conducted by Yang et al. ( 68 ), Chang et al. ( 61 ), de Wispelaere et al. ( 67 ), Mutoh et al. ( 119 ), and Saini and Vrati ( 48 )—demonstrated that VLPs, even without adjuvants, could induce neutralizing antibodies against JEV. In contrast, subunit vaccines typically require adjuvants and booster doses to elicit an adequate immune response. The reviewed articles also indicated that mice immunized with sub-viral particles, with or without adjuvants, developed neutralizing antibodies against JEV ( 53 , 65 ). Section title: Biopolymer based JE NP vaccines Educational score: 4.191065311431885 Domain: biomedical Document type: Study Language: en Chitosan is a positively charged, biocompatible polymer that acts as a natural mucoadhesive agent. As a result, over the past decade, chitosan-derived nanoparticles (CS NPs) have gained widespread use for delivering vaccine antigens via the mucosal route ( 120 ). CS NPs enhance immune activation by promoting the uptake of antigens by DCs and macrophages, which, in turn, stimulates TLR-mediated signaling and the production of pro-inflammatory cytokines ( 121 ). In studies focused on NP vaccine development against JEV, chitosan facilitated the administration of live attenuated vaccines via the I.N. route and the DNA of prM and E protein genes via the I.D. route. The findings revealed that I.N. administration resulted in significantly higher levels of specific anti-JEV IgA. However, cytokine levels and neutralizing antibodies were markedly lower compared to those achieved via S.C. administration ( 83 ). In the case of I.D. delivery of DNA, specific antibodies were generated, conferring 100% and 50% protection with and without adjuvant, respectively ( 69 ). Section title: Biopolymer based JE NP vaccines Educational score: 4.248287200927734 Domain: biomedical Document type: Study Language: en Lumazine synthase (LS) is a family of enzymes that plays a crucial role in versatile vaccine delivery systems due to its oligomeric structure, which exhibits remarkable conformational stability. Lumazine synthase nanoparticles, with their self-assembling properties, facilitate the presentation of antigens to B cells and CD4+ T-helper cells, inducing antibody production and T cell responses ( 122 ). The antigens are displayed in a well-organized array on the surface of LS, creating a high local concentration of antigens. These repetitive patterns facilitate the cross-linking of B-cell receptors through an avidity effect, leading to robust immune responses ( 123 ). A study by Yao and colleagues ( 63 ) demonstrated that LS-assembled EDIII JEV protein significantly increased neutralizing antibody titers (IgG1 and IgG2a) and cytokine levels (IFN-α, IL-12, and IFN-γ). The antibodies elicited by EDIII-LS were comparable to those produced by the live attenuated vaccine (SA14-14-2) and substantially higher than those from the EDIII subunit vaccine. Both EDIII-LS and SA14-14-2 vaccinations achieved 100% protection in challenged mice, whereas only 55% of mice vaccinated with the EDIII subunit vaccine were protected. Section title: Biopolymer based JE NP vaccines Educational score: 4.262633323669434 Domain: biomedical Document type: Study Language: en AB5 toxins are critical virulence factors for major bacterial pathogens, consisting of a catalytic A-subunit that disrupts host functions and a B-subunit that binds to specific glycan receptors on target cell surfaces. The non-toxic B5 component of the holo-toxin (AB5) provides a pentameric scaffold for assembling antigenic proteins, mimicking the native five-fold axis ( 124 ). AB5 toxin-based nanoparticles exploit the unique ability of the AB5 toxin to bind to ganglioside receptors on host cells, leading to enhanced antigen uptake by APCs, triggering both humoral and cellular immune responses ( 125 ). Ahn et al. hypothesized that genetically fusing the B-subunit of the AB5 toxin with a viral antigenic protein would facilitate pentameric self-assembly while preserving the conformational epitopes necessary for an effective immune response. Their study led to the development of a pentameric nanoscale JEV EDIII protein vaccine using cholera toxin B (CTB) and heat-labile enterotoxin B (LTB). The results indicated that both CTB-EDIII and LTB-EDIII recombinant proteins induced high total IgG levels and similar IgG1 levels compared to the inactivated vaccine, as well as comparable neutralizing antibody titers ( 60 ). Section title: Biopolymer based JE NP vaccines Educational score: 4.183284759521484 Domain: biomedical Document type: Study Language: en Bio-nanocapsules, derived from bacterial Nano cellulose, also promote antigen delivery to dendritic cells (DCs), activating CD8+ cytotoxic T cells and enhancing the cross-presentation of antigens, which is crucial for effective cellular immunity ( 126 ). A bio-nanocapsule NP-based anti-JE candidate vaccine was constructed by loading the JEV E protein domain 3 onto the surface of two tandem repeats of the Z domain (ZZ-BNC) derived from Staphylococcus aureus protein A. However, the protection conferred against lethal JEV challenges in immunized mice was relatively low (44.4% without adjuvant and 70% with alum adjuvant), while the inactivated JE vaccine provided complete protection ( 127 ). Section title: Biopolymer based JE NP vaccines Educational score: 4.1616997718811035 Domain: biomedical Document type: Study Language: en Poly (γ-glutamic acid) (γ-PGA) is a biopolymer composed of repeating units of D- and L-glutamic acid, naturally polymerized via γ-amide bonds. γ-PGA possesses excellent biocompatibility and stability, which enhance the adjuvant properties of vaccines by improving immune cell recruitment and antigen persistence, leading to strong long-term immunity ( 128 ). It is non-toxic and biodegradable, allowing for easy uptake by DCs, which leads to cytokine secretion that enhances Th1 immune responses and boosts cytotoxic T lymphocyte (CTL) activity ( 96 ). Okamoto et al. evaluated the adjuvanticity of γ-PGA NPs in combination with an inactivated JEV vaccine, demonstrating that a single dose of the JE vaccine with γ-PGA NPs significantly enhanced neutralizing antibody titers. This resulted in all immunized mice surviving a normally lethal JEV infection, whereas only 50% of those receiving a single dose of the JE vaccine without γ-PGA NPs survived ( 55 ). Section title: Synthetic and inorganic NP-based JE vaccines Educational score: 4.673786640167236 Domain: biomedical Document type: Review Language: en Lipid nanoparticles (LNPs) represent a groundbreaking class of nanoparticles with immense potential for the delivery of nucleic acids and eliciting robust immune responses. LNPs are efficiently taken up by antigen-presenting cells (APCs) via endocytosis or phagocytosis, leading to the release of the encapsulated antigen in the endosome, where it is processed and presented on major histocompatibility complex (MHC) class I and II molecules. This stimulates both CD8+ cytotoxic T cells and CD4+ helper T cells ( 129 ). The antigen presentation activates the adaptive immune response, leading to the production of neutralizing antibodies by B cells and the elimination of infected cells by CD8+ T cells. Additionally, LNPs interact with pattern recognition receptors (PRRs), such as TLR7 and TLR8, on APCs, which recognize viral RNA. This interaction triggers the production of inflammatory cytokines, including IL-6, TNF-α, and type I interferons, enhancing APC maturation and promoting a stronger immune response ( 130 ). Furthermore, the particulate nature of LNPs serves as an adjuvant, boosting the immune response by mimicking viral membranes, which improves both the delivery of antigens and the activation of immune cells ( 131 ). LNPs also facilitate the formation of memory B cells and T cells, ensuring long-term immunity and protection upon re-exposure to the pathogen ( 130 ). This combination of efficient antigen delivery, activation of both innate and adaptive immunity, and adjuvant effects makes LNPs highly effective in vaccines, particularly for viral infections ( 132 ). Section title: Synthetic and inorganic NP-based JE vaccines Educational score: 4.0772552490234375 Domain: biomedical Document type: Study Language: en The promise of LNPs has been underscored by the recent emergency use authorization (EUA) granted by the US FDA for two mRNA-based SARS-CoV-2 vaccines: mRNA-1273 (Moderna) and BNT162b2 (Pfizer-BioNTech). Consequently, researchers involved in vaccine development have become increasingly interested in the mRNA-LNP vaccine platform ( 129 ). Wide arrays of mRNA-encapsulated LNPs are currently under investigation in clinical settings for various applications, including hereditary disorders, viral infections, and cancer ( 28 , 133 ). In a similar vein, Chen et al. developed an mRNA vaccine encoding the JEV prM and E proteins, rigorously evaluating its immunogenicity and protective efficacy. Their findings demonstrated that mRNA immunization could elicit robust JEV-neutralizing antibodies and potent CD8+ T-cell responses, effectively safeguarding mice against JEV infection ( 62 ). Section title: Synthetic and inorganic NP-based JE vaccines Educational score: 4.341342926025391 Domain: biomedical Document type: Study Language: en Polyethylene glycol (PEG) is a versatile polyether molecule that is non-ionic and has multiple applications within the pharmaceutical industry. PEG is commonly used to modify the surface characteristics of nanoparticles (NPs) and enhance their molecular weight (MW) ( 134 ). PEG improves the bioavailability of antigens by reducing clearance through the reticuloendothelial system (RES), allowing for more efficient delivery to APCs. Once internalized, PEG NPs promote the activation of PRRs, including TLRs, on the surface of APCs. This triggers intracellular signaling pathways, such as NF-κB and MAPK, leading to the release of pro-inflammatory cytokines like IL-1β, IL-6, and TNF-α, which help recruit additional immune cells to the site of antigen presentation ( 135 , 136 ). After processing the antigen, APCs present it on MHC class I and II molecules, activating CD8+ cytotoxic T cells and CD4+ helper T cells, respectively. PEGylation also serves as an adjuvant, improving the overall immune response by enhancing antigen delivery, stabilizing the vaccine components, and providing controlled release ( 137 ). Moreover, PEG coating can mitigate immune responses against the nanoparticles themselves, which could otherwise reduce the efficacy of repeated vaccinations ( 136 , 138 ). Hunt et al. utilized PEG to precipitate purified recombinant JEV protein particles. Immunogenicity evaluations revealed that PEG-precipitated recombinant proteins, in conjunction with Freund’s incomplete adjuvant, induced higher neutralizing antibody titers against JEV ( 57 ). Section title: Synthetic and inorganic NP-based JE vaccines Educational score: 4.46064567565918 Domain: biomedical Document type: Study Language: en Well-functionalized gold nanoparticles (AuNPs) are among the most promising nanomaterials for the next generation of vaccines ( 134 , 139 ). Their reliable surface functionalization, biocompatibility, customizable size and shape, and unique optical properties have generated considerable interest in the field of vaccinology. Upon administration, AuNPs are taken up by immune cells, including DCs, macrophages, and B cells, via receptor-mediated endocytosis. The nanoparticles are then transported to lymph nodes, where they present antigens to T cells, initiating the adaptive immune response ( 88 , 140 ). AuNPs also interact with immune cells through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), which activate the innate immune system and trigger the release of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6). These cytokines recruit additional immune cells and create a pro-inflammatory environment that supports adaptive immune activation ( 88 ). Furthermore, gold nanoparticles act as adjuvants, enhancing the immune response to co-administered antigens by increasing both the strength and duration of the response ( 88 , 141 , 143 ). AuNPs also stimulate the production of type I interferons (IFN-α/β) and promote the activation of B cells, CD4+ helper T cells, and CD8+ cytotoxic T cells, which are critical for both humoral and cell-mediated immunity ( 139 , 142 ). Section title: Synthetic and inorganic NP-based JE vaccines Educational score: 4.183582305908203 Domain: biomedical Document type: Study Language: en Nucleic acid strands can be covalently bonded to the cores of gold nanoparticles (typically 13–15 nm) through thiol moieties ( 144 – 146 ). This innovative strategy applies to both DNA and siRNA, which can be directly conjugated to gold cores or to polymer-modified gold cores ( 147 ). Two decades ago, Zhao and colleagues conducted a JE immunization experiment using colloidal gold to inoculate plasmid DNA encoding the prM and E proteins. It remains unclear whether the gold served primarily as a carrier or as an adjuvant in this case. Nevertheless, this immunization approach facilitated a more rapid production of specific anti-JEV neutralizing antibodies via intravenous (I.V.) and intramuscular (I.M.) routes compared to alternative methods. Both active and passive (anti-JEV sera) immunization conferred 100% protection against JEV challenges at 10 5 LD50 ( 47 ). Section title: Conclusion Educational score: 4.046375751495361 Domain: biomedical Document type: Review Language: en Epidemiological data indicate that JEV is steadily expanding into new territories across Europe and Africa, in addition to its established presence in the Asia-Pacific region, with the potential for global dissemination in the near future ( 7 ). While the widespread use of traditional vaccines has successfully reduced the incidence of JE among children in endemic areas, there has been a noticeable rise in cases among adults ( 148 , 149 ). JEV also poses significant threats to animal health and the economy, as it not only causes illness and mortality in livestock but also leads to substantial financial losses for farmers due to reduced productivity. As a result, there is an ongoing demand for effective and safe vaccines that can be produced at scale with minimal financial investment. In response, scientists are actively exploring innovative approaches to develop novel JE vaccines. This review highlights the growing array of nanoparticle-based vaccine candidates that have emerged from the dedicated efforts of researchers. In the foreseeable future, these promising candidates could pave the way for next-generation JE vaccines for both humans and animals as they progress through subsequent stages of the vaccine development pipeline. Section title: Limitations Educational score: 3.943040132522583 Domain: biomedical Document type: Review Language: en The quality of the studies included in this review varied considerably, with some exhibiting methodological weaknesses that could impact the robustness of the findings. The heterogeneity among the studies—particularly regarding administration systems (route, dose, booster immunization, and use of adjuvants) and the methods for evaluating immunogenicity—may limit the comparability of results and their interpretation. Additionally, the review was confined to studies published in English, which may have excluded valuable research published in other languages that could offer further insights. Section title: Future directions for JEV vaccine development Educational score: 3.9277312755584717 Domain: biomedical Document type: Review Language: en Future research on JEV vaccines should address key challenges to improve efficacy and broader applicability. A major focus should be developing broad-spectrum vaccines that provide cross-protection against all five JEV genotypes. Enhancing antigen presentation systems and optimizing antigen combinations could also boost vaccine effectiveness. Additionally, exploring cross-protection against other flaviviruses and improving immunization strategies for broader flavivirus threats will be critical. Tackling these issues will be essential for advancing JEV vaccine development and preparing for emerging flavivirus-related diseases. | Study | biomedical | en | 0.999997 |
PMC11695418 | Section title: Introduction Educational score: 4.042301654815674 Domain: biomedical Document type: Study Language: en The increasing prevalence of multidrug-resistant (MDR) bacterial strains, coupled with the sluggish advancement in the development of novel antibiotics, has rekindled significant interest in the application of phage therapy for the treatment of bacterial infections within clinical practice ( 1–3 ). Bacteriophages, or bacterial phages, are host-specific viruses that parasitize bacteria and replicate by utilizing the host’s metabolic pathways, ultimately resulting in the host’s death. These phages demonstrate a specificity for host infection that operates independently of bacterial antibiotic resistance mechanisms, enabling them to infect and lyse even antibiotic-resistant superbugs ( 4 , 5 ). Section title: Introduction Educational score: 4.187714576721191 Domain: biomedical Document type: Study Language: en Previous studies have demonstrated the potential efficacy of phage therapy in addressing infections caused by drug-resistant bacteria. For instance, the intravenous administration of phages into abscess cavities has been shown to reverse deteriorating conditions and inhibit or eradicate infections caused by multidrug-resistant Acinetobacter baumannii (MDRAB) ( 6 ). Additionally, another study documented the successful treatment of a patient infected with MDRAB through a combined approach involving antibiotics, intravenous phage injections, and aerosolized phage therapy ( 7 ). Several studies have documented the potential of phage therapy in addressing infections caused by multi-drug resistant bacteria ( 8–12 ). However, the extant literature predominantly emphasizes the clinical outcomes in patients, with relatively few investigations examining the biodistribution of phages within the host organism. For instance, in a study where phage therapy was administered to mice via the pulmonary route, phages were subsequently detected in the bloodstream ( 13 ). Similarly, another study involving the inhalation of phages by mice also reported the presence of phages in the serum ( 14 ). However, in a study investigating inhaled bacteriophage therapy using a porcine model of pneumonia, no infectious phages were detected in the serum ( 15 ). Furthermore, most clinical reports on inhaled phage therapy have not examined the distribution of phages within the bloodstream. Specifically, a study on bacteriophages reported an absence of bacteriophages in six blood samples ( 16 ). Although some studies have partially evaluated the distribution of phages, it remains uncertain whether inhaled phages have the potential to enter the bloodstream. Section title: Introduction Educational score: 4.533718585968018 Domain: biomedical Document type: Study Language: en Different routes of administration lead to varying distributions of phages within tissues and distinct elimination processes. Bacteriophages that traverse the digestive tract initially encounter the acidic milieu of the stomach, presenting a significant barrier to their survival. Subsequently, within the intestines, the resident gut microbiota and the intestinal immune system actively surveil and eliminate phage-infected bacteria, thereby indirectly facilitating the clearance of the phages ( 17 ). Bacteriophages that enter the respiratory tract are initially subjected to the mucus-ciliary clearance mechanism, which facilitates the transport of foreign particles, including phages, towards the throat for expulsion. In the lower respiratory tract and lungs, immune cells such as macrophages play a crucial role in phagocytosing and eliminating infected bacteria, thereby restricting the dissemination of phages within the host organism ( 17 ). Additionally, the immune system is integral to the clearance of bacteriophages from the human body. Even in the absence of a specific immune response to phages, macrophages, monocytes, and phagocytes are actively involved in this process ( 18 ). Phagocytes also present phage antigens via antigen-presenting cells. The reticuloendothelial system (RES) of the spleen and liver serves as the primary immune mechanism responsible for phage clearance, effectively reducing phage concentrations to clinically useful levels ( 19 ). A study suggests that B cells are implicated in the clearance of bacteriophages, likely through phage-specific interactions ( 20 ). Additionally, there is evidence indicating that the kidneys also contribute to phage clearance, albeit to a lesser degree. Section title: Introduction Educational score: 3.980524778366089 Domain: biomedical Document type: Study Language: en Numerous case reports have documented the application of phage therapy via inhalation; however, these studies have not examined the impact of inhaled phage therapy on the intestinal microbiota ( 21 ). In a study investigating bacteriophage therapy for Escherichia coli infection in rabbits, researchers observed that oral bacteriophage administration effectively treated intestinal infections while exerting minimal impact on the cecal microbiota ( 22 ). Experimental data indicated that oral phage administration altered the diversity of the gut microbiota ( 23 ). However, the overall impact of oral phage administration on gut bacteria remains inconclusive. Additionally, the effects of inhaled phage therapy on intestinal bacteria have yet to be determined. Further study is necessary to elucidate the effects of phage therapy on the human microbiota ( 24 ). Section title: Introduction Educational score: 3.7164487838745117 Domain: biomedical Document type: Study Language: en In this study, we reported a patient with a pulmonary infection caused by extensively drug-resistant Acinetobacter baumannii (XDRAB) who underwent phage therapy. Additionally, we assessed the presence of inhaled phages in the patient’s biological samples and examined the effects of phage therapy on the patient’s gut microbiota. Section title: Phage screening and preparation Educational score: 4.0705952644348145 Domain: biomedical Document type: Study Language: en The XDRAB strain, isolated from a sputum sample, was utilized for phage screening at the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences ( 25 ). Phage BA3, which demonstrated significant specific lytic activity against XDRAB, was selected for further study. The phage preparation was subsequently purified using a cesium chloride density gradient, followed by dialysis with a Spectra/Por6 membrane in SM buffer (excluding Tris-HCl) to eliminate residual cesium chloride. The phages were then sterilized through filtration with 0.22 μm filters, and the purified phage preparation was stored at 4°C until required for subsequent applications. Section title: Sample collection and DNA extraction Educational score: 4.077569961547852 Domain: biomedical Document type: Study Language: en Sputum, plasma, and fecal samples were collected in sterile tubes in accordance with standard clinical procedures and subsequently stored at −80°C. DNA from sputum samples and cell-free DNA (cfDNA) from plasma samples were extracted using the PathoXtract ® Basic Pathogen Nucleic Acid Kit and the PathoXtract ® Cell-Free Nucleic Acid Kit , respectively, following the manufacturer’s protocols. Total DNA from fecal samples was extracted using the E.Z.N.A. ® Soil DNA Kit (Omega Bio-tek, Norcross, GA, United States). Section title: qPCR assay Educational score: 4.146890640258789 Domain: biomedical Document type: Study Language: en Acinetobacter baumannii was quantified utilizing the A. baumannii Probes-Based Fluorescent Quantitative PCR Assay Kit (CS15-520) from Shanghai C-reagent Biotechnology Co., Ltd. The reaction mixture comprised 12.5 μL of 2× Universal Master Mix (Life Technology), 200 nM of both forward and reverse primers, 100 nM of probes, and 4 μL of template DNA, with water added to achieve a final reaction volume of 25 μL. The primers employed for the identification of A. baumannii phage were AB-pair1-F (5′-GCCATTCGACCATGCGTTAC-3′) and AB-pair1-R (5′-GTCGGATAAAAGCGAACCGC-3′). The reaction mixture comprised 12.5 μL of 2× SYBR GREEN UNIVERSAL MASTER MIX , 200 nM of each primer, and 4 μL of template DNA. The volume was adjusted to a final reaction volume of 25 μL by adding water. qPCR was conducted using the Life Technologies ABI 7500 System platform. The thermal cycling conditions were as follows: initial activation of TaqMan at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 s, and annealing/extension at 60°C for 60 s. The undetectable level of fluorescence was set at a cycle threshold (Ct) of 45. Section title: Amplicon sequencing and data processing Educational score: 4.272126197814941 Domain: biomedical Document type: Study Language: en The hypervariable V3–V4 region of the bacterial 16S rRNA gene was amplified using primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR reaction mixture comprised 4 μL of 5× Fast Pfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of Fast Pfu polymerase, 10 ng of template DNA, and ddH 2 O to achieve a final volume of 20 μL. The PCR amplification cycling conditions were as follows: an initial denaturation at 95°C for 3 min, followed by 27 cycles consisting of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. This was followed by a final extension at 72°C for 5 min, with the reaction terminating at 4°C. In the second round of PCR, index sequences were appended to the termini of the amplicons produced during the initial PCR using primers from the Nextera XT Index Kit (Illumina Inc., San Diego, CA, United States). The PCR amplification was conducted with the following reaction mixture: 10 μL of H 2 O, 20 μL of 5Prime Hot Master Mix, 5 μL of 1 μM forward primer, 5 μL of 1 μM reverse primer, and 10 μL of template pool at a concentration of 1 ng/μL. The amplification protocol employed was as follows: an initial denaturation at 94°C for 3 min; followed by 8 cycles of 94°C for 10 s, 58°C for 30 s, and 72°C for 45 s; and a final extension at 72°C for 10 min. Section title: Amplicon sequencing and data processing Educational score: 4.26287317276001 Domain: biomedical Document type: Study Language: en Purified amplicons were combined in equimolar concentrations and subjected to paired-end sequencing using the Illumina NovaSeq PE250 platform (Illumina, San Diego, United States). The resulting sequences were analyzed utilizing the plugin tools provided within the Quantitative Insights Into Microbial Ecology (QIIME2) bioinformatics package . Two FASTQ files per sample (demultiplexed, paired-end reads) were imported into the QIIME2 environment. The DADA2 denoise-paired plugin was employed to: (i) trim primer sequences and low-quality bases at the read ends, (ii) join paired-end reads, (iii) discard chimeras, and (iv) infer amplicon sequence variants (ASVs). Additional chimera filtering was conducted using the VSEARCH uchime-denovo plugin. ASVs with fewer sequences than 0.005% of the total number of sequences and those not present in at least two samples were subsequently discarded. Taxonomic classification was performed utilizing the feature-classifier classify-sklearn plugin in conjunction with a Naïve Bayes classifier that had been pre-trained on the comprehensive Greengenes 13_8 99% OTU reference database (accessible at http://qiime2.org ). ASVs identified as mitochondria, chloroplasts, or archaea were excluded, along with classifications that were resolved only to the level above phylum. Alpha and beta diversity metrics for each sample were calculated using QIIME with default parameters. Section title: General condition and clinical data Educational score: 4.052244663238525 Domain: clinical Document type: Clinical case Language: en The patient, a 71-year-old female, has a medical history that includes a diagnosis of type 2 diabetes, lower limb venous thrombosis, and severe fatty liver disease. Subsequently, she developed weakness and numbness in the upper limbs, which gradually worsened, along with weakness in the left lower limb. She was diagnosed with ependymoma and underwent surgical treatment. Prior to this hospitalization, the patient experienced fever and cough for 15 days. She had been hospitalized three times for severe pneumonia caused by Acinetobacter baumannii , Nocardia , Pneumocystis jirovecii , and Aspergillus , and had received long-term combination antibiotic treatments. Following hospitalization, she was placed on mechanical ventilation and administered intravenous voriconazole at a dosage of 150 mg every 12 h. A bronchoalveolar lavage fluid (BALF) culture, collected 4 days prior to the initiation of the first phase of phage therapy, tested positive for XDRAB. Following the signing of the informed consent form by the legal representative, the patient received two courses of phage therapy in conjunction with antibiotics. The first phase of phage therapy involved the administration of 0.5 mL × 10 9 PFU/mL via inhalation, twice daily, from January 8, 2022, to January 17, 2022 (a total duration of 10 days). However, 12 days after the completion of the first course of phage therapy, an increased burden of Acinetobacter baumannii was detected in the BALF culture, and Pseudomonas aeruginosa was also cultured. Nevertheless, based on the clinical condition, clinicians did not make further adjustments to the patient’s antibiotic regimen. Then, the patient commenced the second course of phage therapy on January 29, 2022, which continued until February 7, 2022, spanning a total of 10 days. During this phase, the treatment protocol for the initial 3 days entailed the administration of 0.3 mL of 10 10 PFU/mL BA3 phage, diluted in 4.7 mL of saline, delivered via mechanical ventilation twice daily. From the fourth day onward, the regimen was adjusted to administer 0.1 mL of 10 10 PFU/mL phage, diluted in 4.9 mL of saline, via inhalation twice daily. During the two courses of phage therapy, we recorded changes in the patient’s temperature, total white blood cell count, neutrophil percentage, C-reactive protein (CRP), procalcitonin (PCT), and interleukin-6 (IL-6) . Throughout both rounds of phage therapy, the patient’s body temperature remained within normal limits (<37.3°C). Aside from an elevation in the WBC count observed on the seventh day of the first round, the WBC count remained within the normal range. The percentage of neutrophils consistently exceeded 75% during both rounds of therapy. Inflammatory markers, including CRP, PCT, and IL-6, exhibited an increase on the seventh and eighth days of the first round. During the second round of therapy, these inflammatory markers initially increased but subsequently declined . Despite the fluctuations in inflammatory markers observed during the second round of phage therapy, clinicians evaluated the patient’s condition as stable and opted not to implement additional interventions. No severe adverse reactions were noted throughout the course of treatment. Section title: Bacteriophage detection in human samples Educational score: 4.2828593254089355 Domain: biomedical Document type: Study Language: en Phages and pathogen DNA were initially identified in 14 blood samples, 13 sputum samples, and 10 fecal samples collected across two phases of phage treatment using real-time PCR. The relative abundance of the detected pathogens or phages, contingent upon the specific target, is represented by the Ct value. In the blood samples, negative PCR results were predominantly observed for A. baumannii and the bacteriophage DNA, with exceptions noted for the pathogen on day 1 (Ct = 35.8) and day 4 (Ct = 37.8) during the second phase, and for bacteriophages (Ct range: 32.6 to 35.3) within the first 4 days of the second phase . In the sputum samples, there was a gradual increase in the abundance of bacteriophages in the patient’s respiratory tract, as indicated by a decreasing trend in Ct values from 45 to 14.7 during the first phase . Concurrently, the Ct value of A. baumannii exhibited a gradual increase from 13.8 on day 0 to 45 on day 10, indicating a negative result . Unexpectedly, during the second phase, the Ct values of the phage remained between 28.5 and 26.8, while the Ct values of the pathogen, A. baumannii , were relatively lower, ranging from 13.2 to 18.1 . This observation suggests that the phages did not exhibit significant lytic activity against the pathogen in the second phase, thereby highlighting the critical issue of phage resistance in the context of phage therapy ( 26 ). In the fecal samples, the Ct value of the phage on the 10th day post-treatment (22.5) was lower than that prior to the initial treatment (35.5) . This value stabilized at 29 before the commencement of the second phage treatment, subsequently decreasing during the second phase of phage administration (from 32.6 to 22.7) , indicating an increased phage presence in the patient’s gut. Regarding the pathogen, its DNA was detected in only 5 samples from the second phase, exhibiting relatively higher Ct values (34.6–36.5) . This finding suggested that bacteriophages through inhalation may enter and accumulate in the human intestine. Section title: Changes in gut microbiota during phage therapy Educational score: 4.243916034698486 Domain: biomedical Document type: Study Language: en We conducted an analysis of the gut microbiota composition in 10 fecal samples utilizing Illumina-based 16S rRNA sequencing. Our observations indicated that, at a relative abundance threshold of ≥0.05% at the phylum level, there was a substantial increase in the proportion of Proteobacteria (from 49.4 to 95.5%). Concurrently, the relative abundances of the other three dominant phyla— Actinobacteriota , Bacteroidota , and Firmicutes —exhibited varying degrees of reduction during the first phase of phage therapy . Similar to the first phase, Proteobacteria maintained a dominant position; however, the relative abundance of the four predominant phyla— Proteobacteria , Actinobacteriota , Bacteroidota , and Firmicutes —exhibited fluctuations during the second phase of phage application . At the genus level, with a relative abundance of ≥1%, the predominant genera prior to the first round of phage therapy were Acinetobacter (34%), Corynebacterium (27%), and Klebsiella (15%). Following treatment, these shifted to Pseudomonas (48%) and Klebsiella (40%) . Prior to the second phase of phage treatment, Acinetobacter and Pseudomonas constituted 61.9 and 31.7% of the microbial community, respectively. Notably, the genera stricto and Stenotrophomonas exhibited a reduction in abundance to undetectable levels. Conversely, 13 other genera demonstrated an increase in abundance following phage therapy. These genera include Corynebacterium , Bacteroides , Dysgonomonas , Porphyromonas , Elizabethkingia , Lachnoclostridium , gnavus , Veillonella , Achromobacter , Escherichia , Klebsiella , Sphaerochaeta , Pyramidobacter , and Enterococcus . Section title: Discussion Educational score: 3.5735671520233154 Domain: clinical Document type: Clinical case Language: en Following two rounds of phage therapy and continuous clinical antibiotic treatment as part of a comprehensive therapeutic regimen, the patient’s condition stabilized compared to her initial admission. Before discharge, the patient was in a post-tracheotomy state and had successfully been weaned off the ventilator. The patient exhibited no symptoms of fever, abdominal pain, chest tightness, or chest pain, and her mental state and sleep quality were reported to be satisfactory. With medical authorization, the patient was transferred to a rehabilitation hospital for ongoing care. Prior to the patient’s second round of phage therapy, Pseudomonas aeruginosa was also detected. Based on the patient’s condition, clinicians did not adjust the antibiotic treatment regimen. During the second round of phage therapy, the patient’s infection markers (CRP, IL-6) initially increased and then decreased, which may be related to the immune system’s response to Pseudomonas aeruginosa . Section title: Discussion Educational score: 4.340693950653076 Domain: biomedical Document type: Study Language: en In this study, we were unable to detect phages in blood samples during the first round of phage treatment. However, trace amounts of bacteriophage DNA sequences were identified during the second phase of phage therapy. Notably, previous research has detected the presence of bacteriophages in blood samples ( 27 , 28 ). Nevertheless, the question of whether inhaled phages can enter the bloodstream remains a topic of ongoing debate. In alignment with this finding, Chang et al. ( 13 ) identified the presence of phages in the blood of mice following pulmonary administration of phages for the treatment of Pseudomonas aeruginosa pulmonary infections. Similarly, another investigation into phage therapy within a murine model of Pseudomonas aeruginosa infection also documented the presence of bacteriophages in the serum ( 14 ). Conversely, a study examining inhaled bacteriophage therapy in a porcine model of pneumonia did not detect any infectious phages in the serum ( 15 ). Our findings indicate that prolonged inhalation of the phage may facilitate its dissemination into the bloodstream; however, additional clinical trials are necessary to thoroughly assess the safety and implications of this phenomenon. Furthermore, our data demonstrated a progressive decline in phage Ct values in the gut following phage therapy, suggesting an accumulation of phages within the gastrointestinal tract. The mechanisms underlying the distribution of inhaled bacteriophages in the gut, however, remain to be elucidated. Upon inhalation into the lungs, bacteriophages may be translocated to the gastrointestinal tract through internal cellular mechanisms ( 18 ), or alternatively, some inhaled bacteriophages may access the upper digestive tract via the oral cavity, ultimately reaching the intestines ( 17 ). The interactions between bacteriophages and the gut microbiota are notably intricate. The presence of active bacteriophages in the gut may influence the equilibrium of intestinal bacterial populations ( 29 ). Given the limited sample size, we have only performed qPCR detection on bacteriophage DNA in fecal samples. Consequently, we are unable to confirm the presence of viable bacteriophages in the intestine. Assuming the presence of live bacteriophages in the intestinal environment, further research is necessary to elucidate the mechanisms by which inhaled bacteriophages traverse to the intestine. Additionally, it is imperative to assess the safety of bacteriophage inhalation therapy and its potential effects on intestinal microecology. Section title: Discussion Educational score: 4.415329933166504 Domain: biomedical Document type: Study Language: en The composition of a healthy gut microbiota predominantly includes bacteria from four phyla: Firmicutes (60 to 65%), Bacteroidetes (20 to 25%), Proteobacteria (5 to 10%), and Actinobacteria (3%) ( 30 ). However, it is important to consider that the patient’s extended antibiotic treatment may have altered her gut microbiota composition prior to the administration of phage therapy, potentially deviating from the typical profile observed in healthy individuals. Our findings indicate that the predominant bacterial phyla identified were Actinobacteria , Bacteroidetes , Firmicutes , and Proteobacteria , aligning with the composition of a healthy gut microbiota ( 30 ). However, in contrast to a healthy gut microbiota, the patient’s intestines exhibited a marked predominance of the Proteobacteria phylum, ranging from 49.4 to 95.5%. At the genus level, Clostridium constitutes 95% of the phylum Firmicutes within the normal intestinal microbiota, with Bacteroidetes being the subsequent predominant group. In contrast, the patient’s gut microbiota was primarily composed of the genera Acinetobacter , Pseudomonas , and Klebsiella . Notably, the relative abundance of these genera exhibited significant perturbations during the two phases of phage therapy. The influence of bacteriophages on the composition of intestinal microbiota remains a subject of ongoing debate. A study investigating bacteriophage therapy for Staphylococcus aureus device infections reported no significant differences in bacterial abundance within the fecal samples of the patients ( 31 ). In a murine experimental study, researchers observed an increase in the richness and diversity of the microbial flora in the feces of mice treated with phages ( 23 ). Additionally, Febvre et al. ( 32 ) reported that while phage treatment did not induce global changes in the microbiota, it did result in significant alterations in specific microbial community members. Notably, the abundance of E. coli , the target host for the administered phage consortium, was markedly reduced by the conclusion of the treatment period. Section title: Discussion Educational score: 4.220497131347656 Domain: biomedical Document type: Study Language: en Prolonged antibiotic administration significantly impacts the composition of the gut microbiota ( 33 ). The patient, a chronic lung disease sufferer, had been subjected to extended antibiotic treatment prior to admission, which included voriconazole, imipenem, tigecycline, and piperacillin-tazobactam. Antibiotic therapy was maintained post-admission . Consequently, it is anticipated that the gut microbiota experienced disruption due to antibiotic exposure. In this study, a comparative analysis of the gut microbiota before and after phage therapy revealed a reduction in the proportion of the genus Acinetobacter following treatment. Previous research has demonstrated that viruses are capable of translocating across mucosal barriers to reach distal tissues ( 34 ). Inhaled bacteriophages may traverse specific pathways to enter the gastrointestinal tract, potentially altering the composition of the gut microbiota. However, it is important to note that the patient was concurrently administered multiple antibiotics during the course of phage therapy, with adjustments made to the antibiotic regimen based on the patient’s clinical condition. Consequently, the impact of antibiotics on the changes in the patient’s gut microbiota is challenging to quantify or exclude. Our findings, as indicated by Ct values, suggest the accumulation of inhaled phages in the gastrointestinal tract. However, the detection of phage DNA in fecal samples via qPCR does not confirm the presence of viable phages within the gut. Our study was conducted with a single patient, presenting a limited sample size. Consequently, it is challenging to attribute the observed alterations in gut microbiota to the administration of phages based on this isolated case. Further research is warranted to elucidate the potential effects of inhaled bacteriophages on gut microbiota. Section title: Discussion Educational score: 4.2449774742126465 Domain: biomedical Document type: Study Language: en Bacteria can acquire resistance to bacteriophages through multiple mechanisms, including the superinfection exclusion system (Sie) and the CRISPR-Cas system, among others ( 35 , 36 ). Previous study has demonstrated that extended phage therapy can result in the development of phage tolerance ( 26 ). During the first phase of phage therapy in this study, the Ct values of the target bacteria in the patient’s sputum exhibited an upward trend as the treatment advanced. Conversely, the Ct values of the phages demonstrated a decline, indicating successful proliferation of the phages within the bacterial population. This observation suggests a certain degree of efficacy of phage therapy during the first phase of phage treatment. However, during the second round of phage therapy, the Ct values of the target bacteria in the patient’s sputum remained relatively stable and low. Concurrently, the phage Ct values also remained stable but at relatively high levels (28.5–26.8) compared to the first phase. This suggests that the phages did not proliferate effectively within the pathogens during the second phase of treatment. Additionally, Pseudomonas aeruginosa was detected in the patient prior to the second round of phage therapy, which may be related to the patient’s longer hospital stays. Based on these results, we speculate that the extended administration of phages may have contributed to the development of phage tolerance in the patient. Consistent with our findings, Bao et al. ( 37 ) observed the emergence of phage-resistant mutants within a few days during two rounds of phage therapy, highlighting the critical issue of phage resistance in such treatments. Additionally, multiple studies have documented instances of phage therapy where phage-resistant variants were identified ( 6 , 38 , 39 ). Section title: Discussion Educational score: 4.102849006652832 Domain: biomedical Document type: Study Language: en In conclusion, our results suggest the presence of inhaled bacteriophages in human blood and their subsequent accumulation in the intestines. Notably, we observed a stabilization in phage abundance and a sustained high burden of pathogens following the first course of phage treatment. This observation suggests that prolonged phage therapy may induce phage resistance, thereby diminishing its therapeutic efficacy. Our case study also delineates the alterations in relative abundance and diversity at the genus level of the gut microbiome during phage therapy. While numerous studies have indicated that phage therapy may be safe in clinical practice, further clinical trials are necessary to assess the safety and implications of these therapeutic strategies. | Study | biomedical | en | 0.999998 |
PMC11695419 | Section title: 1 Introduction Educational score: 3.861926317214966 Domain: biomedical Document type: Other Language: en Staphylococcus aureus is one of the most significant pathogens in clinical practice, responsible for a wide range of infections in humans, including skin infections, pneumonia, endocarditis, and sepsis . The growing prevalence of drug-resistant strains, particularly methicillin-resistant S. aureus (MRSA), has significantly complicated treatment strategies and is linked to increased mortality rates . Section title: 1 Introduction Educational score: 3.902979612350464 Domain: biomedical Document type: Study Language: en In light of rising antibiotic resistance, increasing attention is being directed toward the study of bacteriophages (phages)—natural bacterial predators. The most promising group of phages for treating staphylococcal infections is the Herelleviridae family, whose members exhibit strictly lytic life cycles and broad host ranges. The efficacy of these phages has been demonstrated in various studies, including in vitro and in vivo experiments, as well as clinical cases . Section title: 1 Introduction Educational score: 3.8050901889801025 Domain: biomedical Document type: Study Language: en Beyond the individual use of phages, there is growing interest in the combined application of phages and antibiotics . Such studies aim to identify optimal combinations and dosages that enhance treatment effectiveness while ensuring safety of application. For instance, the inclusion of phages in therapeutic regimens may reduce the required doses of antibiotics, thereby reducing the risk of side effects. Additionally, combining agents could reduce the likelihood of bacterial resistance development . Section title: 1 Introduction Educational score: 3.9813802242279053 Domain: biomedical Document type: Other Language: en The combined application of antibacterial agents can result in various outcomes, such as additive effects, synergy, or antagonism. In the case of additive effects, the combined action of two agents equals the sum of their individual effects. When the combined efficacy exceeds this additive effect, synergy is achieved. However, when one component limits the other’s activity, resulting in reduced overall antibacterial effectiveness, this is considered antagonism . Section title: 1 Introduction Educational score: 4.027139186859131 Domain: biomedical Document type: Study Language: en In practical applications, it is essential to explore combinations of phages with current antibacterial drugs effective against MRSA. Vancomycin, a glycopeptide antibiotic, is a key first-line treatment for MRSA. However, due to the emergence of resistance , alternative treatments are now used, including linezolid, a synthetic oxazolidinone. Linezolid inhibits protein biosynthesis by binding irreversibly to the 30S and 50S ribosomal subunits, thereby disrupting the formation of the 70S initiation complex and inhibiting peptide chain elongation . Section title: 1 Introduction Educational score: 4.055018901824951 Domain: biomedical Document type: Study Language: en It should be noted that prolonged linezolid use can lead to serious side effects, such as lactic acidosis . Furthermore, the use of linezolid against microorganisms with minimum inhibitory concentrations (MICs) of 4 μg/mL (at which S. aureus is classified as sensitive; bacterium is considered resistant when MIC ≥ 8, according to the Clinical and Laboratory Standards Institute (CLSI) standards) or higher may lead to suboptimal clinical outcomes, potentially due to issues with drug delivery and plasma concentration variability . Section title: 1 Introduction Educational score: 3.896129608154297 Domain: biomedical Document type: Study Language: en The combined use of lytic phages with linezolid, including at sub-inhibitory concentrations, has been proposed as a strategy to improve therapeutic outcomes and mitigate adverse effects. Previous studies have demonstrated a synergistic effect of linezolid and phages on planktonic and biofilm-associated bacterial cells . However, other researchers have raised concerns about combining bacteriostatic antibiotics like linezolid with phages, as the replication of phages might be hindered . These discrepancies have also been observed in animal model experiments . Section title: 1 Introduction Educational score: 4.137714385986328 Domain: biomedical Document type: Study Language: en In this study, we investigated the individual and combined effects of sub-inhibitory concentrations of phage vB_SauM-515A1, a member of the Herelleviridae family, and linezolid on S. aureus . The combined action of these two agents was evaluated against both planktonic cells and biofilms. To further characterize the interaction between the phage and linezolid, transcriptional analysis of the infected bacterial strain was performed. This study aims to expand our understanding of the mechanisms underlying the combined use of lytic phages and antibiotics against S. aureus , which is critical for optimizing therapeutic efficacy. Section title: 2.1 Bacterial strain and bacteriophage Educational score: 4.088953971862793 Domain: biomedical Document type: Study Language: en The previously characterized S. aureus SA0413Rev strain was selected from the strain collection at the Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine . Bacterial cultures were grown in lysogeny broth (LB) (Oxoid, UK) or on LB agar (Oxoid) at 37°C. Susceptibility to linezolid (Sigma-Aldrich, USA) was determined according to the CLSI standards. Multilocus sequence typing (MLST) of the strain was performed following the standard method . Section title: 2.1 Bacterial strain and bacteriophage Educational score: 3.9221999645233154 Domain: biomedical Document type: Study Language: en The lytic phage vB_SauM-515A1, used in this study, belongs to the Kayvirus genus within the Herelleviridae family and exhibits typical myovirus morphology. It was originally isolated from a commercial Staphylococcus bacteriophage cocktail (batch P332) produced by Microgen (Russia), utilizing the S. aureus strain SA515 (spa-type t008, ST8) as the host. This phage has been extensively characterized in previous studies . Section title: 2.1 Bacterial strain and bacteriophage Educational score: 4.115447521209717 Domain: biomedical Document type: Study Language: en For all subsequent experiments, the phage was propagated as follows: an overnight culture of S. aureus SA0413Rev (OD 620 = 0.6) was diluted 1:100 in fresh LB broth and grown to an OD 620 = 0.12. The phage was then added at a multiplicity of infection (MOI) of 0.1, and the culture was incubated at 37°C with shaking (200 rpm) for 24 h. The resulting phage lysate was centrifuged at 10,000 g for 10 min at 4°C to remove bacterial debris. The supernatant was subsequently filtered through a 0.22 μm membrane filter (Merck Millipore, USA) to obtain a sterile phage preparation. Section title: 2.2 Evaluation of phage-antibiotic synergy in planktonic bacterial cultures Educational score: 4.104054927825928 Domain: biomedical Document type: Study Language: en The individual and combined effects of the phage and linezolid were investigated using a checkerboard assay as previously described , with some modifications. Briefly, phage vB_SauM-515A1 was added to the wells of a 96-well flat-bottom polystyrene microplate (Thermo Scientific, USA) containing LB, with final concentrations ranging from 0 to 10 5 PFU/mL (serial 1:10 dilutions) in the horizontal wells. Linezolid was added vertically, with final concentrations ranging from 0 to 32 μg/mL (serial 1:2 dilutions). Bacterial suspensions (OD 620 = 0.12; 1.2 × 10 8 CFU/mL) were added to achieve a final concentration of 10 4 CFU/mL (Colony-forming unit). Positive controls consisted of LB medium inoculated with the bacterial strain, while sterile LB was used for negative controls. The final volume in each well was 200 μL. The phage and antibiotic effects were monitored by measuring optical density at 620 nm every hour for 24 h at 37°C using a Microplate Reader Flex-A (Allsheng, China). All experiments were performed in triplicate. The results were converted to percentage reduction relative to the positive control using the following formula : Section title: 2.2 Evaluation of phage-antibiotic synergy in planktonic bacterial cultures Educational score: 3.8294036388397217 Domain: biomedical Document type: Study Language: en To assess the combination effect, the fractional inhibitory concentration (FIC) index was calculated according to the following formula : Section title: 2.2 Evaluation of phage-antibiotic synergy in planktonic bacterial cultures Educational score: 4.047967910766602 Domain: biomedical Document type: Study Language: en where MIC_antibiotic and MIC_phage represent the MICs of the antibiotic and phage, and C_antibiotic and C_phage are the respective inhibitory concentrations of the antibiotic and phage in combinations. FIC values were interpreted as follows: synergy (FIC < 0.5), additive effect (0.5 ≤ FIC < 2), and antagonism (FIC ≥ 2). Section title: 2.3 Biofilm formation assay Educational score: 4.117640018463135 Domain: biomedical Document type: Study Language: en The biofilm formation assay followed the previously described method with modifications. Bacterial suspensions in the exponential growth phase (OD 620 = 0.12) were inoculated into wells containing tryptic soy broth with 1% glucose (TSBg, Himedia, India) at a final concentration of 10 4 cells per well, and incubated for 48 h at 37°C without shaking. After incubation, the wells were gently washed with sterile phosphate-buffered saline (PBS) to remove planktonic cells and stained with 0.1% crystal violet (Sigma-Aldrich, USA). Sterile medium was used as the negative control. Biofilm formation was quantified by measuring the optical density at 570 nm. All experiments were performed in triplicate. Section title: 2.3 Biofilm formation assay Educational score: 3.9631564617156982 Domain: biomedical Document type: Study Language: en Biofilm formation was classified according to following criteria : no biofilm (OD ≤ OD C ), weak biofilm (OD C < OD ≤ 2 × OD C ), moderate biofilm (2 × OD C < OD ≤ 4 × OD C ), and strong biofilm (OD > 4 × OD C ), where OD C = average OD of the negative control + (3 × SD of the negative control). Section title: 2.4 Evaluation of antibacterial treatments on biofilm associated cells Educational score: 4.089169979095459 Domain: biomedical Document type: Study Language: en The evaluation of antimicrobial treatments on biofilm-associated cells was performed in accordance with the study by Dickey and Perrot , with minor modifications. Biofilms were grown for 48 h and washed with sterile PBS as aforementioned. After washing, 200 μL of TSBg containing various combinations of antibacterial agents were added to each well of a 96-well microplate. Bacteriophage (1 MOI) or linezolid at two concentrations (2 or 10 MIC) was used for individual treatment. For simultaneous treatments, both bacteriophage and linezolid were applied at the corresponding concentrations. The negative control contained TSBg without any antibacterial agents. Incubation was conducted for 48 h at 37°C. For sequential treatment, 200 μL of TSBg with bacteriophage (1 MOI) was added and incubated for 24 h at 37°C. Subsequently, linezolid (2 or 10 MIC) was added in an amount not exceeding 5% of the total volume, followed by an additional 24 h incubation at 37°C. After treatment, biofilms were disrupted by pipetting, and viable cells were determined by serial dilution plating on LB agar, followed by incubation at 37°C for 24 h. All experiments were performed in triplicate. Section title: 2.4 Evaluation of antibacterial treatments on biofilm associated cells Educational score: 3.40412974357605 Domain: biomedical Document type: Study Language: en The interaction effects of the phage and antibiotic were calculated using the coefficient of interaction (COI) as described by Kumaran et al. : Section title: 2.4 Evaluation of antibacterial treatments on biofilm associated cells Educational score: 4.016514301300049 Domain: biomedical Document type: Study Language: en where A R - reduction in bacterial counts by treatment with antibiotic, P R - reduction in bacterial counts by treatment with phage, and AP R - reduction by the combined treatment (staggered or simultaneous). Results were interpreted as synergistic (COI > 0), additive (COI = 0), or antagonistic (COI < 0). A mixed model analysis was performed to assess the significance of COI results, with a P -value < 0.05 considered significant. Section title: 2.5 Biofilm scanning electron microscopy (SEM) imaging Educational score: 4.17254638671875 Domain: biomedical Document type: Study Language: en Biofilms were grown directly on catheters (Apexmed International B.V., Netherlands) placed in a 24-well plate (Corning, USA). Exponentially growing bacterial suspensions (OD 620 = 0.12) were added to the wells containing TSBg medium at a final concentration of 10 4 cells per well. After 5 days of incubation at 37°C, the medium was replaced with fresh TSBg. Antibacterial treatment was carried out as described above in Section 2.4, using bacteriophage at 108 PFU/mL and linezolid at 2 MIC. The samples were then processed for scanning electron microscopy (SEM) imaging as previously described . Briefly, biofilms were fixed with 2.5% glutaraldehyde (Sigma-Aldrich, USA) in PBS for 2 h at room temperature, and then washed with PBS three times. The dehydration was performed using the following series of ethanol–water mixtures: 10, 20, 30, 40, 50, 60, 70, 70, 80, 90, 96, and 96% (12 steps, 5 min each). Next, the samples were chemically dried with a 10 min incubation in HMDS: ethanol 1:1 mixture, two 10 min incubations in 100% HMDS, and one incubation in 100% HMDS until complete evaporation. Preliminary coated with a ∼10 nm gold-palladium alloy layer using Eiko IB 3 ion coater (Japan), the samples were observed with a Zeiss Merlin microscope equipped with Gemini II Electron Optics (Zeiss, Oberkochen, Germany). The images were acquired at low accelerating voltage (2 kV) and low probe current (100 pA) via HE-SE2 detector. The FIJI software (NIH, Bethesda, MD, USA) was used to process SEM images . Section title: 2.6 One-step growth curve of bacteriophage Educational score: 4.114934921264648 Domain: biomedical Document type: Study Language: en The one-step growth curve of phage vB_SauM-515A1 on the S. aureus strain SA0413Rev was performed as previously described , with the addition of a sub-inhibitory concentration of linezolid (1/4 MIC). Briefly, S. aureus SA0413Rev cells in the early exponential phase (OD 620 = 0.12) were infected with the phage (MOI 0.001) in the presence of linezolid. The mixture was incubated for 7 min at 37°C, followed by centrifugation for 4 min at 10,000 g . The pellet was resuspended in 300 μl of LB. Aliquots of 10 μl were collected at 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, and 120 min post-infection. Samples were treated with 1% chloroform, and the number of phage particles was determined using the double agar overlay assay . A sample without the addition of the antibiotic served as a positive control. All experiments were performed in triplicate. Section title: 2.7 DNA extraction Educational score: 4.0728678703308105 Domain: biomedical Document type: Study Language: en Genomic DNA was isolated from an overnight culture of S. aureus SA0413Rev (OD 620 = 0.6) grown in LB broth using the Wizard Genomic DNA Purification Kit (Promega, USA) following the manufacturer’s protocol. DNA concentration and quality were assessed using a Qubit 4 Fluorometer and Nanodrop ND-1000 (Thermo Fisher Scientific). Section title: 2.8 Illumina library preparation and sequencing Educational score: 4.09636116027832 Domain: biomedical Document type: Study Language: en A total of 100 ng of isolated DNA was used for library preparation with the KAPA HyperPlus Kit (Roche, Switzerland), following the manufacturer’s protocol. The library was subjected to final cleanup using KAPA HyperPure Beads (Roche, Switzerland). Library size distribution and quality were assessed using a High Sensitivity DNA chip (Agilent Technologies), and quantification was performed using the Quant-iT DNA Assay Kit, High Sensitivity (Thermo Fisher Scientific). Section title: 2.8 Illumina library preparation and sequencing Educational score: 3.7512974739074707 Domain: biomedical Document type: Study Language: en The DNA libraries were sequenced on the HiSeq 2500 platform (Illumina, USA) according to the manufacturer’s instructions. The following reagent kits were used: HiSeq Rapid PE Cluster Kit v2, HiSeq Rapid SBS Kit v2 (200 cycles), and HiSeq Rapid PE FlowCell v2, along with a 2% PhiX spike-in control. Section title: 2.9 Oxford nanopore library preparation and sequencing Educational score: 4.1226019859313965 Domain: biomedical Document type: Study Language: en Libraries were prepared according to the manufacturer’s protocol using NEB reagents, with long reads generated on the PromethION platform (Oxford Nanopore Technologies, UK). The sequencing libraries were prepared with the ligation sequencing kit SQK-LSK109 and the native barcoding expansion kit EXP-NBD196, and run on an R9.4.1 (FLO-PRO002) flow cell. Basecalling was performed using Guppy v6.5.7 with default parameters (high accuracy model, minimum quality score ≥ 7). Section title: 2.10 Total RNA extraction Educational score: 4.062155723571777 Domain: biomedical Document type: Study Language: en For transcriptomic analysis, S. aureus cultures (OD 620 = 0.12) were treated with either the phage, linezolid, or a combination of both. The antibiotic was used at a concentration of 1/4 MIC, while the phage was added at an MOI of 10. Untreated cultures served as controls. At each time point (5, 20, 30, and 50 min after exposure to the antibacterial agents), 1 mL of the culture was collected, centrifuged (3 min at 5,000 g ) at 4°C, and the cell pellets were immediately frozen at −70°C. All experiments were performed in triplicate. Section title: 2.10 Total RNA extraction Educational score: 4.139614105224609 Domain: biomedical Document type: Study Language: en RNA was extracted from the frozen cell pellets using the MagMAX mirVana Total RNA Isolation Kit (Thermo Fisher Scientific, Lithuania) on the KingFisher Flex Purification System (Thermo Fisher Scientific, USA) following the manufacturer’s protocol. RNA was treated with DNase using the Turbo DNA-Free Kit (Thermo Fisher Scientific) in a 50 μl volume and further purified using Agencourt RNAClean XP beads (Beckman Coulter, USA). The total RNA concentration was measured with the Quant-iT Ribogreen RNA Assay Kit (Thermo Fisher Scientific), and the quality of the extracted RNA was verified using an Agilent Bioanalyzer with RNA 6000 Pico Chips (Agilent Technologies, USA). Section title: 2.11 RNA library preparation and sequencing Educational score: 4.150086402893066 Domain: biomedical Document type: Study Language: en For transcriptomic library preparation, 150 ng of total RNA was used as input. Ribosomal RNA was selectively removed using the Ribo-Zero Plus rRNA Depletion Kit (Illumina, USA), followed by library preparation with the KAPA RNA Hyper Kit (Roche, Switzerland) according to the manufacturer’s protocol. RNA purification steps involved the use of RNA Clean XP magnetic beads (Beckman Coulter, Brea, USA), and final library cleanup was done with Agencourt AMPure XP beads (Beckman Coulter, Brea, USA). Library size distribution and quality were assessed using an Agilent High Sensitivity DNA kit (Agilent Technologies, USA), and library concentration was determined using the Quant-iT DNA Assay Kit, High Sensitivity (Thermo Fisher Scientific, USA). Section title: 2.11 RNA library preparation and sequencing Educational score: 4.023936748504639 Domain: biomedical Document type: Study Language: en Equimolar amounts of all libraries (12 pM) were sequenced on the Illumina HiSeq platform using 2 × 100 bp paired-end reads with a 5% PhiX spike-in control. RNA-seq read data were deposited in the NCBI Sequence Read Archive under bioproject PRJNA1172966 . Section title: 2.12.1 Genome assembly Educational score: 4.3759236335754395 Domain: biomedical Document type: Study Language: en Taxonomic confirmation of the sequenced reads was accomplished with Kraken2 v2.1.2 and Bracken v2.8 . The quality assessment of short paired-end reads was performed using falco v1.2.1 and MultiQC v1.17 . Adapters removal and reads filtering was performed using fastp v0.23.4 . Long-reads quality was evaluated with Nanoq v0.10.0 . Prior to assembly, long reads were filtered with Filtlong v0.2.1 1 . Hybrid assembly was created using Unicycler v0.5.0 , Medaka v. 1.11.3 2 , MaSuRCA v. 4.1.0 , and Polypolish v. 0.6.0 . PGAP 2023-10-03.build7061 was used for annotating the assembly. Minimap2 v2.26-r1175 was used to map long reads to the assembly. BWA MEM v0.7.17-r1188 was employed for mapping short reads to the assembly. Subsequently, SAMtools v1.17 and mosdepth v0.3.5 were used to compile mapping statistics. Prophage sequences were identified using the PHASTEST web server . Antibiotic resistance genes and resistance-conferring mutations were detected with ResFinder v.4.1 . Virulence Finder 2.0 was employed to search for virulence genes, with an identity threshold of 90% and a minimum protein length of 60% 3 . The genome of the S. aureus SA0413Rev has been deposited in GenBank under accession number CP173176 . Section title: 2.12.2 Differential gene expression analysis Educational score: 4.2514495849609375 Domain: biomedical Document type: Study Language: en Sequenced reads were aligned to the reference S. aureus SA0413Rev genome using STAR v2.7.11a . SAMtools v1.17 software was utilized for sorting and converting SAM files to BAM format, followed by indexing and subsequent statistical analysis. Mapping quality and coverage along genes were evaluated with QualiMap v2.2.2 , and individual reports were merged using MultiQC v1.17. Reads were assigned to genes using featureCounts v2.0.6 . Differential gene expression analysis utilized the edgeR v3.42.4 package for R. Genes with a false discovery rate (FDR) cutoff of 0.05 and a fold change (log 2 FC) threshold of | 1| (i.e., ≥| 2| -fold change) were deemed differentially expressed. For gene ontology (GO) enrichment analysis, GO categories were annotated using the PANNZER2 tool with a Positive Predictive Value (PPV) cutoff of 0.5. Gene ontology (GO) enrichment analysis was conducted using GOpiscator v1.0.5 4 . Section title: 2.12.3 Statistical analysis and data visualization Educational score: 1.5808004140853882 Domain: biomedical Document type: Other Language: en Figures were created using R v4.3.0 using the following packages: ggplot2 v3.5.1 , cowplot v1.1.1 , ggnewscale v0.5.0.9000 5 , ggh4x v0.2.8 6 , colorspace v2.1-0 , scales v1.3.0 , gridtext v0.5.1 7 , and ComplexHeatmap v2.16.0 . For statistical analyses R packages rstatix v0.7.2 , DescTools v0.99.50 , and Python 3.11 module statsmodels v 0.13.5 8 were used. Section title: 3.1 Overview of experimental strategy Educational score: 4.1210503578186035 Domain: biomedical Document type: Study Language: en To compare the effects of individual and combined antimicrobial agents, we used the phage vB_SauM-515A1 and linezolid, a key antibiotic against MRSA. The characteristics of S. aureus strain SA0413Rev and phage vB_SauM-515A1 have been described previously . This strain was sensitive to both the phage and linezolid, belonged to the clinically relevant sequence type ST8, and exhibited spa type t008, as well as multidrug resistance. According to the biofilm formation assay, SA0413Rev was classified as a moderate biofilm producer. Section title: 3.1 Overview of experimental strategy Educational score: 4.0914483070373535 Domain: biomedical Document type: Study Language: en To assess the effects of individual and combined antimicrobial agents on planktonic cells of S. aureus SA0413Rev, we employed the checkerboard assay, measuring cell growth dynamics over 24 h. The nature of the combined effect was evaluated using the fractional inhibitory concentration (FIC) index. Section title: 3.1 Overview of experimental strategy Educational score: 4.062023162841797 Domain: biomedical Document type: Study Language: en Since biofilm formation is a critical factor in the pathogenesis of S. aureus infections, we also investigated the ability of the strain to form biofilms and evaluated the impact of individual and combined antibacterial agents on biofilm-associated cells. We tested different treatment regimens, including the simultaneous administration of both agents and sequential treatment (phage followed by antibiotic). The results were interpreted using the coefficient of interaction (COI). Additionally, scanning electron microscopy was employed to visualize structural changes in the biofilms. Section title: 3.1 Overview of experimental strategy Educational score: 3.423748254776001 Domain: biomedical Document type: Study Language: en Based on one-step growth curves, we selected time points for the final stage of the study—evaluating the bacterial transcriptional response to the combined treatment. The complete genome of the SA0413Rev strain was sequenced and characterized ( Supplementary Text ). Section title: 3.2 Effect of individual and combined treatment with phage and linezolid on planktonic cells of S. aureus Educational score: 4.080337047576904 Domain: biomedical Document type: Study Language: en The evaluation of individual antimicrobial agents revealed that the minimum MOI for the phage and the MIC for linezolid that completely inhibited bacterial growth were 0.1 and 4 μg/mL, respectively . Lower concentrations of the antimicrobial agents slowed bacterial growth compared to the control but did not achieve complete inhibition. Section title: 3.2 Effect of individual and combined treatment with phage and linezolid on planktonic cells of S. aureus Educational score: 4.141787528991699 Domain: biomedical Document type: Study Language: en Under combined treatment with sub-inhibitory concentrations (MOI 0.001 and 0.01 for the phage, and 1/8 to 1/2 MIC for linezolid), significant inhibition of bacterial growth was observed, with more than a 90% reduction in culture growth . The FIC index for the combination was 0.225, indicating a synergistic effect. For instance, the combination of 1/8 MIC of linezolid and 0.01 MOI of the phage completely suppressed bacterial growth . Section title: 3.3 Effect of phage and linezolid on S. aureus biofilms Educational score: 4.116550922393799 Domain: biomedical Document type: Study Language: en The effect of antibacterial agents on biofilm-associated cells was evaluated based on CFU counts, which demonstrated a synergistic interaction when phage vB_SauM-515A1 was applied simultaneously with linezolid at concentrations of 2 MIC and 10 MIC ( p < 0.05; COI > 0) . The individual application of linezolid at both concentrations showed moderate efficacy in reducing the number of viable cells, whereas the phage exhibited even lower activity under the same conditions. Sequential treatment of pre-formed biofilms with phage vB_SauM-515A1 followed by linezolid, regardless of the concentration, resulted in an antagonistic effect ( p < 0.05; COI < 0). Notably, complete eradication of bacteria was not observed with any treatment method, while the most effective combination achieved only ∼4-log reduction. Section title: 3.3 Effect of phage and linezolid on S. aureus biofilms Educational score: 4.101530075073242 Domain: biomedical Document type: Study Language: en SEM was employed to visualize the structural effects of the antimicrobial agents and their combination (phage MOI 1 and linezolid concentration 2 MIC) on S. aureus biofilm formed on a catheter over 5 days . The extended growth period was chosen to mimic clinical scenarios, where biofilms typically form on medical devices over longer periods. As result, neither individual treatment with linezolid or phage , nor their sequential application , led to a noticeable reduction in the biofilm compared to the untreated control . In contrast, simultaneous application of both agents resulted in a visible reduction of the biofilm matrix, with individual bacterial cells appearing in the field of view . Section title: 3.4 Transcriptional response of S. aureus to individual and combined exposure to phage vB_SauM-515A1 and linezolid Educational score: 4.089483737945557 Domain: biomedical Document type: Study Language: en The transcriptional response of S. aureus to antimicrobial agents was examined at 5, 20, 30, and 50-min time points, selected based on the growth curves of the phage’s lytic cycle . Compared to the uninfected control, the combined treatment of strain SA0413Rev with the phage and antibiotic resulted in 839 differentially expressed genes (DEGs) ( Supplementary Table 1 ). For cultures treated individually with linezolid or phage, the number of DEGs was 651 and 680, respectively. Section title: 3.4 Transcriptional response of S. aureus to individual and combined exposure to phage vB_SauM-515A1 and linezolid Educational score: 3.9783880710601807 Domain: biomedical Document type: Study Language: en Analysis of DEG distribution by agent and time point revealed that the highest number of DEGs was detected in the later stages of the experiment for any type of treatment . It was also found that, in the combined treatment case, the phage contributed the most to the transcriptional response. Common DEGs across all time points suggested a non-specific cellular response to both the individual and combined treatments. Section title: 3.4 Transcriptional response of S. aureus to individual and combined exposure to phage vB_SauM-515A1 and linezolid Educational score: 4.103792667388916 Domain: biomedical Document type: Study Language: en Functional analysis of the DEGs using gene ontology (GO) identified enrichment across all major categories: biological processes (BP), molecular functions (MF), and cellular components (CC) ( Supplementary Table 5 ). A more detailed examination of the BP category showed that transcriptional changes in enriched GO groups were mostly unidirectional across all time points and within each category ( Supplementary Tables 1 , 2 ). Section title: 3.4 Transcriptional response of S. aureus to individual and combined exposure to phage vB_SauM-515A1 and linezolid Educational score: 4.315109729766846 Domain: biomedical Document type: Study Language: en Phage treatment at the 5-min time point resulted in decreased transcription of genes involved in nitrate assimilation , glycerol catabolic process , maltodextrin transmembrane transport , D-tagatose 6-phosphate catabolic process , phosphoenolpyruvate-dependent sugar phosphotransferase system , tricarboxylic acid cycle , isoleucine biosynthetic process , and carbohydrate phosphorylation . Upregulated genes were mainly associated with pyrimidine biosynthesis . At 20 and 30 min, all GO categories included only upregulated genes, representing processes such as amino acid metabolism , cytolysis in another organism , prophage region activation , programmed cell death , and cytolysis . At 30 min, amino acid metabolism , cytolysis in another organism , and prophage region activation were again detected, with similar categories observed at 50 min, except those related to amino acid metabolism. Genes related to programmed cell death and phosphate ion transport were also upregulated at 50 min. Transcription levels of genes responsible for nickel cation transport were downregulated, except for genes encoding phosphate ABC transporter permease subunits PstA and PstC. Section title: 3.4 Transcriptional response of S. aureus to individual and combined exposure to phage vB_SauM-515A1 and linezolid Educational score: 4.29389762878418 Domain: biomedical Document type: Study Language: en Upon linezolid treatment at 5 min, only two enriched GO groups were detected: ‘ de novo ’ IMP biosynthetic process with upregulated genes, and maltodextrin transmembrane transport with downregulated genes. At 20 min, most genes with increased transcription were also associated with the ‘ de novo ’ IMP biosynthetic process , with a smaller portion involved in nitrate assimilation . In contrast, genes involved in the riboflavin biosynthetic process were downregulated. At the 30-min time point, two categories with downregulated genes were detected: ‘ de novo ’ IMP biosynthetic process and translation . The GO category associated with cell adhesion contained upregulated genes, except for those encoding the metal ABC transporter substrate-binding protein and zinc ABC transporter substrate-binding lipoprotein AdcA. The term related to nickel cation transport contained both upregulated and downregulated genes, while genes involved in the small molecule metabolic process were exclusively downregulated. The final time point, 50 min, similarly included ‘ de novo ’ IMP biosynthetic process and translation , and also identified the proteolysis category , mostly with upregulated genes, except for those encoding Abi family protein, signal peptidase II, and zinc metalloproteinase aureolysin. Section title: 3.4 Transcriptional response of S. aureus to individual and combined exposure to phage vB_SauM-515A1 and linezolid Educational score: 4.224310874938965 Domain: biomedical Document type: Study Language: en Under combined treatment with both phage and linezolid, at the 5-min time point, genes associated with pyrimidine nucleotide biosynthesis were upregulated, while nitrate assimilation , tricarboxylic acid cycle , and maltodextrin transmembrane transport were downregulated. At 20 min, upregulated genes were found in groups related to cytolysis in another organism and programmed cell death , while only one downregulated group was identified—glycine decarboxylation via the glycine cleavage system . The 30-min time point showed a similar set of upregulated categories, including cytolysis in another organism , programmed cell death , and viral tail assembly . Genes with reduced transcription at 30 min were observed in the tricarboxylic acid cycle and lactose metabolic process . The final 50-min time point included only upregulated genes, associated with processes such as cytolysis in another organism , nitrate assimilation , and prophage region activation . Section title: 4 Discussion Educational score: 4.0916595458984375 Domain: biomedical Document type: Study Language: en In this study, we conducted a comprehensive investigation into the combined effect of linezolid and the lytic phage vB_SauM-515A1 against S. aureus SA0413Rev. This strain belongs to the highly prevalent clonal complex CC8, one of the most widespread clones both in Russia and globally , known for causing hospital-acquired infections. The selected phage is a classical representative of the Herelleviridae family, considered one of the most promising for therapeutic use . Section title: 4 Discussion Educational score: 4.2814130783081055 Domain: biomedical Document type: Study Language: en Our results demonstrate that the combination of phage vB_SauM-515A1 and linezolid against planktonic S. aureus SA0413Rev cells led to a synergistic effect. Similar findings of synergistic interactions between antimicrobial agents against S. aureus have been previously demonstrated using the checkerboard assay, showing synergy between sub-inhibitory concentrations of linezolid and a phage cocktail that included a member of the Herelleviridae family . Conversely, an antagonistic effect was observed with the combination of another phage, PYOSa ( Herelleviridae ), and linezolid . Through CFU and PFU counts before and after treatment with antibacterial agents (over 24 h), the authors showed that bacteriostatic antibiotics, including linezolid, inhibited the complete lysis of bacterial cells, regardless of the order of treatment. Moreover, final concentration of phage particles decreased with simultaneous treatment of antibiotics. Notably, in that study, linezolid was used at a super-MIC concentration of 10 μg/mL, likely critically limiting the availability of the bacterial translational machinery for phage propagation. A similar result was observed, where linezolid at the same concentration (10 μg/mL) limited the amplification of a phage mixture that included bacteriophage K ( Herelleviridae ) . Section title: 4 Discussion Educational score: 4.118198394775391 Domain: biomedical Document type: Study Language: en In our study, we also observed a negative effect of sub-inhibitory linezolid concentrations on phage replication, reflected by a prolonged latent period (from 20 to 30) and an overall extension of the phage life cycle (from 60 to 100) . However, this limitation did not significantly impact the lytic activity of phage vB_SauM-515A1 . In contrast, it was previously demonstrated that sub-inhibitory linezolid concentrations (1 μg/mL) shortened the adsorption time and latent period while increasing progeny release for phage MR-5 ( Herelleviridae ) . Section title: 4 Discussion Educational score: 3.9715895652770996 Domain: biomedical Document type: Study Language: en In summary, linezolid does affect the phage life cycle, with concentration likely playing a crucial role in the overall effect on planktonic cells. Additionally, the observed differences could be attributed to variations in pre-treatment times with the antibiotic and the specific interactions between the phage and the bacterial strain. Section title: 4 Discussion Educational score: 4.22077751159668 Domain: biomedical Document type: Study Language: en The study of the ability of antimicrobial agents to eliminate biofilm and the bacterial cells within it, as presented here, confirmed the importance of considering the sequence of agent administration—a point highlighted in several studies . In our case, a sequential application led to antagonism, while synergy was observed with simultaneous administration. Synergy from the simultaneous use of phage PYO ( Herelleviridae ) and various antibiotics, including linezolid (2 MIC), against S. aureus strain Newman was also reported . Notably, in that case, synergy was also observed with sequential administration, aligning with other studies . These discrepancies could be explained by strain-specific differences in S. aureus . One possible factor is the variation in biofilm formation capacity between strains. The S. aureus Newman strain forms weak biofilms , whereas the S. aureus SA0413Rev strain is a moderate biofilm producer, which may influence the resulting effect. Additionally, the choice of phage may play a role, particularly its ability to disrupt biofilm integrity, thereby increasing permeability to antibiotics . It appears that phage vB_SauM-515A1, when used alone, is not effective in targeting biofilms, as confirmed by our SEM results. Section title: 4 Discussion Educational score: 4.014843940734863 Domain: biomedical Document type: Study Language: en It is worth noting that, despite the observed synergistic effects in our study, complete elimination of cells from biofilms was not achieved. This finding aligns with previous research and highlights the ongoing challenges in combating biofilm-associated infections . A promising approach to address this issue could be the combination of phages and/or antibiotics with agents that degrade the biofilm matrix, such as phage depolymerases. Such a strategy has the potential to enhance treatment efficacy by improving the accessibility of antimicrobial agents to bacterial cells . Section title: 4 Discussion Educational score: 4.01899528503418 Domain: biomedical Document type: Study Language: en To further investigate the mechanisms underlying the combined action of these antibacterial agents, we performed transcriptional analysis. This approach has been widely applied in phage-bacteria interaction studies, including those focusing on Herelleviridae staphylophages . The transcriptional profile of phage vB_SauM-515A1 used in this study has been previously characterized in detail , along with the host strain SA515 (ST8) response to phage infection . Section title: 4 Discussion Educational score: 4.15957498550415 Domain: biomedical Document type: Study Language: en In this study, transcriptional profiles were obtained for bacteria treated individually with linezolid, bacteriophage, or their combination. The presence of shared DEGs across all conditions suggests a nonspecific cellular response to the antimicrobial agents. This response involved a reduction in the transcription of genes associated with the utilization of alternative carbon sources, such as maltodextrin. Changes were also observed in energy metabolism, specifically nitrate assimilation, including the transcription of genes encoding parts of the nitrate reductase complex ( narI , narJ ) and nitrite reductase ( nirB ). During the early stages of infection, these genes were downregulated under phage treatment but upregulated with linezolid. In the combined treatment, transcription levels of the nitrate assimilation genes initially decreased and then increased, likely reflecting bacterial attempts to compensate for energy deficits to enhance survival. Section title: 4 Discussion Educational score: 4.142050743103027 Domain: biomedical Document type: Study Language: en In the case of the combined treatment, most DEGs reflected the effect of the phage , indicating a typical productive phage infection. This was characterized by alterations in pyrimidine biosynthesis, virulence-related genes transcription, and prophage region activity, consistent with previous findings . Specifically, we observed upregulation of leukocidin genes located within prophage regions as well as hemolysin genes located outside these regions . Section title: 4 Discussion Educational score: 4.167346000671387 Domain: biomedical Document type: Study Language: en Differences were also observed in the transcriptional response of S. aureus SA0413Rev to phage infection, both with and without the antibiotic, compared to the host strain SA515’s response to the same phage . In the host strain, transcription of genes associated with nucleotide metabolism was reduced, while in SA0413Rev, as well as in S. aureus SH1000 and S. aureus Newman infected with phage K ( Herelleviridae ), these genes were upregulated . Additionally, changes in amino acid metabolism were detected in SA515’s transcriptional response to phage infection, but no significant changes were observed when phage and linezolid were applied together . Section title: 4 Discussion Educational score: 4.2589111328125 Domain: biomedical Document type: Study Language: en Treatment of S. aureus SA0413Rev with phage vB_SauM-515A1, both with and without linezolid, led to increased transcription of the genes encoding a holin-like protein CidA and an antiholin-like proteins LrgA and LrgB. In contrast, phage infection in the host strain only elevated transcription of LrgB . These proteins play key roles in biofilm formation and maintenance, as they regulate the balance between cell lysis and survival . During cell lysis, extracellular DNA is released, becoming part of the biofilm matrix and thereby strengthening its structure . This may explain why prior phage treatment restricts linezolid access to biofilm cells, contributing to the observed antagonism during sequential treatment. These variations highlight the strain-specific nature of bacterial responses to antimicrobial agents. Section title: 4 Discussion Educational score: 4.261776447296143 Domain: biomedical Document type: Study Language: en In the combined treatment, two unique enriched categories were identified: glycine decarboxylation via the glycine cleavage system (GCS) and lactose metabolism. Transcription levels of the genes in these categories were reduced, except for the YafY family transcriptional regulator gene. The suppression of these processes can have significant negative effects on the overall cellular metabolism and fitness. The GCS is closely linked to one-carbon metabolism, which is essential for nucleotide and amino acid synthesis. Limiting access to alternative carbon sources, such as lactose, reduces the ability of bacteria to generate energy, leading to slower growth. Thus, the simultaneous action of the phage and linezolid induces a broader metabolic imbalance by restricting the synthesis of key metabolites and energy production. Section title: 4 Discussion Educational score: 4.238358020782471 Domain: biomedical Document type: Study Language: en Analysis of the strain’s transcriptional response to linezolid, in addition to the nonspecific response described above, revealed changes in translation , specifically upregulation of ribosomal protein genes, consistent with linezolid’s known mechanism of action . During combined treatment, transcription of genes encoding both large and small ribosomal subunit proteins also changed, though the GO term for translation was not significantly enriched. Additionally, following 30 min of antibiotic exposure, there was increased transcription of genes encoding the MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), which play a crucial role in S. aureus biofilm formation and overall virulence by promoting adhesion, colonization, and immune evasion . Section title: 4 Discussion Educational score: 4.189302921295166 Domain: biomedical Document type: Study Language: en Here, we reported a comprehensive evaluation of the combined effect of phage vB_SauM-515A1 and linezolid on the S. aureus strain SA0413Rev. This combination approach requires careful selection of antibacterial agent conditions (concentration, administration order, phage type, and antibiotic) to avoid potential negative outcomes, such as antagonistic effects. This consideration is important for both planktonic cells and biofilms, though it is particularly applicable to planktonic cells due to the absence of antagonism. Our analysis of the transcriptional response of SA0413Rev revealed significant changes in several essential biological processes. At all time points, the influence of both antibacterial agents was identified, with the phage contributing a larger impact, likely due to its high concentration (10 MOI). It is common practice to use high phage concentrations in studies of bacterial transcriptional responses to phage infection to ensure synchronized infection . However, this approach may impose limitations on studying combined treatments. On the other hand, using lower phage concentrations might result in insufficient phage particles to detect a bacterial response. Validation of this approach requires further investigation in future studies. | Other | biomedical | en | 0.999998 |
PMC11695421 | Section title: Introduction Educational score: 3.81064772605896 Domain: biomedical Document type: Review Language: en Globally, cardiometabolic multimorbidity (CMM) referring to the coexistence of two or more cardiometabolic diseases (CMDs), including diabetes, hypertension, coronary heart disease (CHD) and stroke, is closely related to decreased quality of life and increased economic burden of disease ( 1–3 ). Section title: Introduction Educational score: 4.143980979919434 Domain: biomedical Document type: Study Language: en Currently, scientists became interested in new risk factors for CMDs. Here, plasma homocysteine (Hcy), a sulfur-containing amino acid, an intermediate product of methionine metabolism, has been identified as a newly discovered potential risk factor for a variety of diseases including diabetes, hypertension, neurodegenerative diseases, osteoporosis, and cancer ( 4 ). Its mechanisms refer to inflammatory response and oxidative stress, vascular endothelial cells damage, prothrombotic, pro-smooth muscle cell proliferation, methylation, and lipid metabolism disorder in vivo ( 5 ). Numerous epidemiological studies have established the associations between an increase in plasma Hcy levels and CMDs. For instance, an 11-year follow-up study from NHANES found that Hcy was a promising biomarker in risk stratification among diabetic patients ( 4 ). Additionally, it has been shown that 75% of hypertension cases also have hyper-Hcy (HHcy), called H-type hypertension, which was first introduced by Chinese researcher in 2008 ( 6 , 7 ). The risk of CVDs in hypertension patients with HHcy was approximately 5 and 12 times that of single-hypertension and healthy people, respectively ( 8 ), and HHcy was independently associated with atherosclerotic plaques and stroke ( 9 ). Although the associations between Hcy and single CMDs are well-established, a limited number of studies examined the associations between Hcy and CMM. So far, only one study has reported an association between Hcy and CMM, while no positive result has been found ( 10 ). Section title: Introduction Educational score: 4.1193695068359375 Domain: biomedical Document type: Study Language: en Studies have confirmed that the generation of Hcy is completely dependent on the methionine cycle metabolism pathway, and human methionine is completely obtained from food, so the influence of diet on HCY cannot be ignored ( 11 ). The association between Hcy and CMM will be differed by confounders level (such as diet and lifestyles, etc.) in different directions and magnitudes. Thereby, a case–control study based on propensity-score-matched method was conducted in this study, using a semiparametric approach to increase the likelihood of a reasonable match between the case and the control group, and dealing with multiple confounders or stratification, greatly improving the reliability of the results ( 12 ). Additionally, the prevalence of HHcy is much higher among adults in China than in other countries ( 13–15 ). And most of epidemiological studies only focused on hospital-based populations, with few based on general community populations, which may limit interpretation of these data. Those were mainly concentrated in North China and rare in South China ( 14 ). Thus, we conducted a community-based, propensity-score-matched, and case–control study to obtain the real-word evidence of the association between Hcy and CMM by investigating the multi-aspects influencing factors of CMM, to determine the common biomarkers of CMM, and to provide a scientific basis for the preventive and therapeutic strategies of CMM in Chinese adults. Section title: Study design and sample Educational score: 4.083458423614502 Domain: biomedical Document type: Study Language: en In this study, firstly, we conducted a population-based cross-sectional study to explore the association of Hcy with CMM, then, a 1:1 matched case–control study was conducted using propensity-score-matched method to validate this association. A representative sample was obtained by multistage cluster random sampling design from July 2013 to March 2014 in Hunan Province (including 14 districts and a population of more than 66 million), China. Detailed information on study design of this population has been described in our previous study ( 16 ). Those met the inclusion and exclusion criteria signed written informed consent. After obtaining informed consent, eligible participants were asked to complete a questionnaire. Accordingly, 4,012 participants were included in the population-based cross-sectional study to explore the relationship between Hcy and CMM. Furtherly, a 1:1 matched case–control study based on the propensity-score-matched method (414 No-CMM and 414 CMM) was conducted to verify this effect . The matched factors include age, sex, educational attainment, family income, occupation status, marital status, current smoking, heavy alcohol consumption, unhealthy diet, inactive exercise, sedentary behavior, BMI, WC, TC, TG, LDL-C and HDL-C because each factor individually contributes to increased risk of cardiometabolic diseases according to previous researches ( 16 , 17 ). Section title: Study design and sample Educational score: 4.18055534362793 Domain: biomedical Document type: Study Language: en The sample size estimation formula for cross-sectional studies was as follow: n = u α 2 p 1 − p δ 2 , here, δ and p represents the estimated allowable error and population rate π , respectively. According to the previous study, the prevalence of HHcy in the population is 35.4% ( 18 ), with an error requirement of no more than 3%, if We assume α = 0.05, then, the sample size obtained is 977. A total of 4,012 subjects were included in this study, which meet sample size needs. The test power was 1.000 based on the prevalence of HHcy (58.3and 32.2%, respectively) in the CMM group (436 participants) and non-CMM group (3,576 participants). In addition, the sample size estimation formula for 1:1 case–control study was as follows: m = Z 1 − a / 2 / 2 + Z β P 1 − P 2 P − 0.5 2 , here, m refers to the number of pairs with inconsistent exposure status between the case and control, and P = OR / 1 + OR . Next, calculate the total number of required pairs (M): M = m P 0 1 − P 1 + P 1 1 − P 0 , here, P 0 and P 1 are estimated exposure rates for the control group and the case group, respectively. Based on previous literature reports, the HHcy exposure rate in the control group was 66.4%, OR = 2.08 ( 19 ), and the setting α = 0.05 (bilateral), β = 0.20, and the final estimated sample size was 127 pairs. The case and control groups in this study included 414, respectively, which meet the sample size needs. The test power was 1.000 based on the prevalence of HHcy in the case and control groups (57.2 and 36.7%, respectively). Section title: Data collection Educational score: 4.067991256713867 Domain: biomedical Document type: Study Language: en Well-trained investigators collected data on socioeconomic characteristics through face-to-face and one-to-one questionnaires, which was referred the questionnaire of the China Health and Retirement Longitudinal Study (CHARLS) ( 20 ), including demographic characteristics (including age, sex, education, family income, occupation status and marital status) and lifestyles factor (including smoking, alcohol drinking, exercise and diet). Here, a test–retest reliability test was performed on the questionnaire, with a Cronbach’s α coefficient in this sample being 0.778. Education level: below of high school, ordinary/vocational high school and undergraduate/college degree. Family income per year was asked for every participant, and further divided into three groups: low, medium and high. Marital status groups: unmarried, married/cohabitation and divorce/widow. Occupation types: wage-laborer, white-collar worker, farmer and retiree. Smoking was defined in the questionnaire as smoking more than 100 cigarettes in life. The drink frequency (days) and amounts (drinks) in the past week were reported according to a set of simple and easy-to-understand photos to measure the drinks of different kinds of drinks . The average daily alcohol drinks were estimated as follows: average daily alcohol drinks = (frequency [days] × (amounts [drinks] in each of those days))/7 ( 18 ). Heavy alcohol consumption level was defined as average daily alcohol drinks ≥2. We evaluated dietary status using food frequency questionnaire according to a more recent dietary recommendation for blood pressure and combining with traditional Chinese eating habits, which considered adequate consumption of fresh fruit, fresh vegetables, unprocessed meats (including red meat, fish or shellfish), reduced consumption of high-fat, high-salt and sugar-sweetened food. We defined an unhealthy diet as meeting less than four items of the recommendations (see Supplementary methods for details). For physical activity, the number of days per week that the participants did physical activities (such as weightlifting, stair climbing, fast cycling, aerobics, running, etc.) and the time of each exercise were obtained through the questionnaires. Inactive exercise was defined as having lasting for less than 10 continuous minutes per week ( 21 ). Sedentary behavior was defined as sitting still for more than 3 hours per day. Section title: Anthropometric measurement Educational score: 4.039283752441406 Domain: biomedical Document type: Study Language: en Systolic and diastolic blood pressure was measured by professionally trained staff using the calibrated electronic automatic tester. Each participant had their blood pressure measured three times with at least 5 min of rest each time. The average of the 3 values was calculated and documented as the final blood pressure value. Weight, height, and waist circumference (WC) were measured by trained staff using well-calibrated instruments. Body-mass index (BMI) was calculated as bodyweight in kg divided by the square of height in meters, and a value more than or equal to 24 was defined as an abnormal BMI. Section title: Biochemical measurement Educational score: 4.019000053405762 Domain: biomedical Document type: Study Language: en Blood samples were collected at 07:30–10:00 after a fasting period of 12 h. The plasma Hcy was measured by trained laboratory technicians using the microplate enzyme immunoassay method, with homocysteine Detection Kit of MedicalSystem Biotechnology Co., Ningbo, China . Other laboratory indicators included fasting blood-glucose (FPG), plasma total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) and C-reactive protein (CRP) were detected using a Hitachi 7,600 Automatic Biochemistry Analyzer (Hitachi). Section title: Definition of cardiometabolic diseases Educational score: 4.009653091430664 Domain: biomedical Document type: Study Language: en Among cardiometabolic diseases, we focus on the diabetes, hypertension, coronary heart disease, and stroke. Diabetes mellitus was defined as a fasting blood glucose level ≥ 7.0 μmol/L or clinically diagnosed with diabetes mellitus or taking hypoglycemic drugs. Hypertension was defined as a diastolic/systolic blood pressure ≥ 90/140 mm/Hg or clinically diagnosed with hypertension or taking antihypertensive drugs. Coronary heart disease and stroke could be defined as a self-reported disease diagnosed by a physician or hospitalization record. Cardiometabolic multimorbidity was defined as the coexistence of two or three cardiometabolic diseases, including hypertension, diabetes, coronary heart disease and stroke. Section title: Statistical analysis Educational score: 3.9187772274017334 Domain: biomedical Document type: Study Language: en The mean and standard deviation were used to describe the normal distribution of quantitative variables, and the t test was used to compare the differences. The median and quartile intervals were used to represent the quantitative variables that did not follow the normal distribution, and the Wilcoxon rank sum test was used to compare the differences. Categorical variables were expressed as counts and percentages (%), and differences were compared using Chi-square test or Fisher exact probability method. Section title: Statistical analysis Educational score: 4.055742263793945 Domain: biomedical Document type: Study Language: en Participants were categorized into two groups based on the number of CMM: ≥2 and none. Quartile classifications of Hcy level was generated (first quartile [Q1]: < 10.4 μmol/L, second quartile[Q2]: 10.4 ~ 13.2 μmol/L, third quartile[Q3]:13.3 ~ 16.2 μmol/L, fourth quartile [Q4]: >16.2 μmol/L). Logistic regression models were employed to evaluate the associations between Hcy and CMM using odds ratios (ORs) and 95% confidence intervals (CIs). The potential non-linear relationships of Hcy with CMM was estimated by using a restricted cubic spline (RCS) model. “VennDiagram” and “openxlsx” R packages were used to implement multimorbidity pattern analysis. Section title: Statistical analysis Educational score: 4.095952987670898 Domain: biomedical Document type: Study Language: en Here, to validate the association of Hcy with CMM, a 1:1 matched case–control study was established using propensity score matching method. The data before and after matching were, respectively, compared to see whether the patients were balanced in important covariates. The difference of the covariates after matching was greater than 0.06, indicating that the covariates after matching were balanced among groups (see Supplementary Table S1 for details). In this case–control study, to examine the proportion of CMM in the exposed population that theoretically would not have occurred if all participants had adhered to the lowest level of Hcy (Q1), we calculated the attributable risk proportion (ARP) under the assumption of a causal relationship between the highest level of Hcy (Q4) and CMM risk. The formula for calculating ARP was as follows: ARP = (OR−1)/OR×100%. Section title: Statistical analysis Educational score: 4.085453987121582 Domain: biomedical Document type: Study Language: en To identify the potential mechanism for the association, we conducted a mediation effect model as Hcy (X) → biomarker (M) → CMM(Y). A biomarker was selected if it was significantly correlated with both Hcy and CMM. Detailly, a logistic model was used when CMM was the dependent variable, and a linear model was used when the biomarker was the dependent variable. Bias-corrected bootstrap method with 1,000 resamples was used to obtain 95% CIs of the direct and indirect effects ( 22 ). Age, sex, educational attainment, family income, occupation status, marital status, current smoking, heavy alcohol consumption, unhealthy diet, inactive exercise and sedentary behavior were adjusted in mediation models as covariates. Coefficients are presented in standardized form, using standardized coefficients as indices of effect. A statistically significant mediation effect is observed when the 95%CI does not include zero. The mediated proportion was used to evaluate the effect size of the mediation analysis. Section title: Statistical analysis Educational score: 3.7663493156433105 Domain: biomedical Document type: Study Language: en To assess the robustness of the results, four models were constructed in the sensitivity analysis based on previous researches ( 16 , 18 ). In model 1, no covariate was adjusted. In model 2, we adjusted for age, sex, education, family income, marital status and occupational status. In model 3, sedentary behavior should be added as an adjusted variable. In model 4, additionally adjusted for serum FPG, TC, TG, LDL-C and HDL-C and CRP based on Model 3. In addition, subgroup analyses were performed according to different combinations of CMMs. Section title: Statistical analysis Educational score: 1.9242626428604126 Domain: biomedical Document type: Study Language: en All statistical analyses were conducted using STATA version 18.0 (Institute, Gary, NC, United States) and R version 4.4.1. A two-sided p < 0.05 was considered statistically significant. Section title: Population characteristics Educational score: 4.085650444030762 Domain: biomedical Document type: Study Language: en A total of 4,012 participants were enrolled in this study for analysis ( Table 1 ). The study population had a mean age of 54.6 years (SD 12.6). 1,644 (41.0%) were males and 2,368 (59.0%) were females. Among them, 2,179 (54.3%) of 4,012 individuals did not have a CMD, whereas 1833 (45.7%) had at least one of these diseases. The prevalence rate of diabetes, hypertension, CHD and stroke were 12.2% , 34.8% , 7.7% , and 2.8% , respectively. The prevalence of CMM was 10.9% . Compared with people who did not have CMM, those with CMM were more likely to be older, male, have low educational attainment and family income, be farmers or retirees, be currently smoking, heavy drinking, unhealthy in diet, sedentary and overweight/obesity, and have diabetes, hypertension, stroke and CHD ( Table 1 ). The CMM group had elevated mean levels of WC, FPG, SBP, DBP, TG, TC and CRP (88.9 ± 10.6, 7.5 ± 2.4, 153.7 ± 20.5, 87.5 ± 13.7, 2.4 ± 2.0, 4.9 ± 1.0, and 6.1 ± 2.4 mmol/L, respectively) than the No-CMM group. The prevalence rate of diabetes, hypertension, CHD and stroke were 12.2% , 34.8% , 7.7% , and 2.8% , respectively. Notably, those with CMM were more likely to have high level of plasma Hcy than population with No-CMM. Section title: Multimorbidity pattern analysis Educational score: 4.099257946014404 Domain: biomedical Document type: Study Language: en Figure 2 provides details about the combinations of different cardiometabolic diseases and includes 15 combinations in total. Among the 4,102 participants enrolled in this study, 1833 had at least one of CMD (45.7%) and 436 (10.9%) met the diagnostic criteria for CMM, which includes metabolic disorders such as hypertension, diabetes mellitus, stroke and CHD. Of those with CMM, the most two frequent combinations were the “Diabetes + Hypertension” and “Hypertension + CHD,” corresponding to 47.5% (207/436) and 26.8% (117/436), respectively. Additionally, among the total study participants 9.2% (40/436) had there or more component conditions of CMM . Here, the associations of plasma Hcy with the risk of specific combination of CMD were displayed in Supplementary Table S2 . After adjusting for age, sex, educational attainment, family income, occupation status, marital status, current smoking, heavy alcohol consumption, unhealthy diet, inactive exercise, sedentary behavior, BMI, WC, TC, TG, LDL-C, HDL-C and CRP, we found those with diabetes, hypertension and CHD had corresponding ORs of 1.97 (95% CI: 1.15–3.56, p = 0.013), 11.35 (95% CI: 5.58–23.09, p < 0.001), and 1.98 (95% CI: 1.05–3.74, p = 0.034), respectively. No significant association was found between plasma Hcy and stroke (OR = 1.00, 95% CI: 0.92–1.05, p = 0.691). Section title: Associations of homocysteine with cardiometabolic multimorbidity and its ARP Educational score: 4.097725868225098 Domain: biomedical Document type: Study Language: en Multiple logistic regression models were carried out to examine the impact of plasma level of Hcy on the risk of CMM . In the cross-sectional study based on 4,012 participants, after adjusted for the covariables (including age, sex, educational attainment, family income, occupation status, marital status, current smoking, heavy alcohol consumption, unhealthy diet, inactive exercise, sedentary behavior, BMI, WC, TC, TG, LDL-C, HDL-C and CRP), compared with the lowest level of plasma Hcy [Q1], the third (OR = 1.51, 95%CI:1.06–2.16, p = 0.023) and fourth quartile levels [Q4] (OR = 2.82, 95%CI: 2.03–3.91, p < 0.001) of Hcy were significantly and positively associated with increased CMM risk. Moreover, RCS analysis showed that there was an inverse dose–response relationship between plasma Hcy concentration and CMM after adjusted for the covariables, suggesting that escalating levels of Hcy was associated with an increasing risk of developing CMM ( p < 0.001) . Section title: Associations of homocysteine with cardiometabolic multimorbidity and its ARP Educational score: 4.099607467651367 Domain: biomedical Document type: Study Language: en In the propensity-score-matched case–control study (414 non-CMM vs. 414 CMM), 828 participants were included. We validated the results of the above cross-sectional study that plasma Hcy had positive impact on risk of CMM ( Table 2 ). For details, compared with those with lowest quartile level of Hcy, the risk of CMM was increased by 183% (OR = 2.83, 95% CI: 1.84–4.36, p < 0.001) among those with highest fourth quartile level of Hcy. Table 2 also summarized the ARP for plasma level of Hcy. In case–control study, the multivariate ARP of highest fourth quartile level of Hcy was 64.66% (95% CI: 46.24–77.06%). Section title: Mediation analysis Educational score: 4.098653793334961 Domain: biomedical Document type: Study Language: en Supplementary Tables S3 , S4 illustrated the correlations of plasma Hcy with various potential mediated indicators and the association of potential mediated indicators with CMM. Since CRP, TC, TG and WC were significantly correlated with both Hcy and CMM, we hypothesis that these indicators may mediate the association we investigated. We performed a causal mediation analysis, and recognized a proportion of effect mediated by CRP, TC, TG and WC as 7.77% (95% CI: 2.87–16.00%, p < 0.001), 12.00% (95% CI: 5.10–21.00%, p < 0.001), 18.93% (95% CI: 8.96–40.00%, p < 0.001) and 22.58% (95% CI: 11.26–56.00%, p < 0.001), respectively . Section title: Sensitivity and subgroup analysis Educational score: 4.053515434265137 Domain: biomedical Document type: Study Language: en In sensitivity analyses, we constructed several models, then, we found the results remained similar in all sensitivity analyses . Moreover, we conducted subgroup analysis. Supplementary Figure S3 showed the results stratified by different combinations of CMM. For details, the plasma level of Hcy was mostly correlated with the combination of diabetes, hypertension and CHD (OR = 2.26, 95%CI: 1.43–3.57, p < 0.001), followed by the combination of diabetes and hypertension (OR = 1.34, 95%CI: 1.23–1.44, p < 0.001), the combination of hypertension and CHD (OR = 1.32, 95%CI: 1.21–1.44, p < 0.001) and the combination of hypertension and stroke (OR = 1.19, 95%CI: 1.04–1.37, p = 0.013), respectively . Section title: Discussion Educational score: 4.0928497314453125 Domain: biomedical Document type: Study Language: en In this community-based study, we investigated the association of Hcy with CMM (two or more conditions in diabetes, hypertension, coronary heart disease, and stroke) in a group of adults over 30 years of age in China, with the largest effect combination of CMM being diabetes, hypertension and coronary heart disease. An inverse association and dose–response relationship were observed between CMM and plasma Hcy levels. Highest level of plasma Hcy (> 16.2 μmol/L) explained about 64.66% of the CMM risk. We also recognized a significant mediation effect by C-reactive protein, total cholesterol, triglyceride and waist circumference in associations of Hcy with CMM, with corresponding mediation proportion ranging from 8 to 23%. Our findings suggested a targeted approach to dynamically monitor and reduce plasma Hcy levels to reduce the risk of cardiometabolic multimorbidity. Section title: Discussion Educational score: 4.103446006774902 Domain: biomedical Document type: Study Language: en Hcy was identified by Butz et al. ( 23 ), as an important intermediate product in the methionine cycle and cysteine metabolism. Subsequently, an 8-year-old intellectually disabled boy died strangely from a severe heart attack, and HHcy’s description was first reported. Since that time, the number of clinical and basic studies of Hcy has increased exponentially. In certain situation, the risks associated with HHcy may be significantly elevated. There is evidence that HHcy is a high predictive value for diabetes patients ( 24 ), hypertension ( 25 ), and cardiovascular and cerebrovascular diseases ( 26 ). In our study, we found high level of plasma Hcy was positively related to elevated risk of diabetes, supporting the result from other study ( 24 ). In addition, Hcy was also associated with increased risk of the occurrence of complications of diabetes, such as diabetic nephropathy, diabetic retinopathy, etc. In the future, Hcy can potentially be used as an early screening indicator for diabetic microvascular complications ( 27 ). Additionally, homocysteine, as one of one-carbon metabolism components, is also closely entwined with gestational diabetes mellitus risk ( 28 ). Therefore, it is very important to understand the origin and metabolism of homocysteine and the pathway of its change in diabetes pathogenesis. Section title: Discussion Educational score: 4.082938194274902 Domain: biomedical Document type: Study Language: en In 2007, a study on the efficacy and safety of enalapril folic acid tablets in lowering blood pressure and plasma Hcy was conducted in six major cities in China ( 6 ). Thus, Chinese researchers first proposed the concept of HTH, and found elevated Hcy levels were associated with 75% of the hypertension cases ( 7 ). Numerous human epidemiological studies have reported the relationship between plasma Hcy levels and hypertension different populations ( 25 , 29 ). In our study, we found those with over 16.2 umol/L plasma Hcy level had a 11.35 times higher risk of hypertension than those in lowest levels (<10.2 umol/L), which was consistent with serval studies ( 25 , 29 , 30 ). Notably, HTH was significantly amplifying the damage of hypertension to blood vessels, increasing the risk of heart, brain, and kidney complications, and increasing the risk of cardiovascular events, especially stroke ( 9 ). Section title: Discussion Educational score: 4.2263078689575195 Domain: biomedical Document type: Study Language: en Since the observation, by McCully et al., of severe arteriosclerotic lesions in children with high level of plasma Hcy, a numerous series of observational studies have demonstrated elevation of plasma Hcy level was considered be related to cardiovascular disease (CVD), stroke, and venous thromboembolism ( 31 , 32 ). Another study suggested that even slight increases in plasma Hcy levels can enhance the risk of CVD ( 33 ). In our study, we recognize the high level of plasma Hcy (> 16.2 μmol/L) plays a key role in prevalence to coronary heart disease (OR = 1.98, p = 0.034). A review reported that primary stroke can at least in part be prevented by lowering total homocysteine. Detailly, total Hcy values in adults of 10 μmol/L or below are probably safe, but that values of 11 μmol/L or above may justify intervention ( 34 ). The strongest evidence that Hcy plays a causal role in atherothrombosis reducing stroke has been provided by studies using animal models ( 35 ). These studies support the role of Hcy in the pathogenesis of atherosclerosis, suggesting that Hcy may be a disease biomarker ( 34 ). However, some studies have proposed different views on the relationship between Hcy and stroke. Recent meta-analysis did not show that reducing Hcy treatment was associated with incidence rate of all-cause death or coronary artery disease, except for prevention for stroke ( 36 ). Therefore, it is still uncertain that whether Hcy is a causative factor or a biomarker of vascular disease in human beings. Further research may be needed to elucidate its causal role and mechanism on vascular disease in different populations. Section title: Discussion Educational score: 4.161716461181641 Domain: biomedical Document type: Study Language: en With the aging of the global population and the development of medical technology, the number of chronic diseases is constantly expanding, and more and more chronic diseases are being controlled at a certain level, resulting in delayed survival time for patients ( 37 ). This has led to an increasing number of people suffering from CMM (co-occurrence of at least two CMDs, including diabetes, hypertension, stroke and coronary heart disease), posing a huge challenge to the healthcare system ( 38 ). It has been confirmed that plasma Hcy is highly correlated with individual CMDs (such as diabetes, hypertension and coronary heart disease), as shown by our present findings, to the best of our knowledge, however, no research has further evaluated the impact of plasma HCY on CMM, except for one research that reported negative result ( 10 ). Our study provides important and innovative results to explore the strong correlation between plasma Hcy and CMM, and dose–response relationship analysis suggests that the higher the plasma Hcy level, the greater the risk of CMM. And, in our sensitivity analysis, the results were not materially changed with those of the main analyses. Notably, our subgroup analysis showed that high level of plasma Hcy was most associated with the combination of coexistence of diabetes, hypertension and CHD among the general population in China. Critically, we calculate the ARP for exposure on high level of plasma Hcy. In case–control study, the multivariate ARP of highest fourth quartile level of Hcy was 64.66% (95% CI: 46.24–77.06%). It means that the percentage of CMM attributable to Hcy in those exposed to the highest levels of plasma Hcy (> 16.2 umol/L) was 64.66%, indicating that 64.66% of those with high levels of Hcy would avoid CMM if they reduced their Hcy levels to the lowest one. Section title: Discussion Educational score: 4.245009422302246 Domain: biomedical Document type: Study Language: en Cardiometabolic disease spectrum has gradually expanded to any metabolic disease that can affect the function and/or structure of the heart, including diabetes, CVD, hypertension, chronic renal insufficiency, etc., forming a chronic disease spectrum characterized by metabolic disorder of multiple organ systems ( 39 ). In terms of mechanism, with the continuous deepening of relevant clinical and basic research, the academic community has found that a series of pathological and physiological changes, such as inflammation, glucose and lipid metabolism disorders, oxidative stress, hormone regulation imbalance, and changes in intracellular and extracellular signaling pathways, are involved in the pathogenesis of CMM ( 40 ). Among them, inflammation damage caused by macrophages which typically exhibit classic M1 polarization, then secrete pro-inflammatory cytokines, participate in inflammatory responses (such as CRP, IL-6, etc.), and promote vascular damage, playing a core role in the development of CMD ( 41 , 42 ). In other hand, lipid metabolism disorder caused by excessive oxidative stress participates in the pathogenesis of CMD ( 40 ). Similarly, data from UK Biobank found that evidence of serum TC and TG are causally related to the risks of cardiometabolic multimorbidity ( 43 ). In our study, cellular inflammatory factor (CRP) and abnormal indicators of lipid metabolism (TC, TG and WC) mediate a small proportion (approximately 8 ~ 23%) of the association of Hcy with CMM, suggesting that the unexplained variations might be attributable to other mechanisms of CMM. CRP and lipids indicators (TC, TG) may serve as early biomarkers of Hcy-related CMM. However, more investigations are warranted to target the clinical heterogeneity of CMM. Section title: Discussion Educational score: 4.17303466796875 Domain: biomedical Document type: Study Language: en This study we conducted has some strengths. To the best of our knowledge, our study provides important and new evidence to this field that those with plasma Hcy levels exceeding 16.2 umol/L have a higher risk of CMM, and confirmed that Hcy was mostly associated with the combination of diabetes, hypertension and CHD, implicating for cardiologists and decision makers that CMM can be attributable to high level of plasma Hcy and it has become a public threat persistently affecting cardiovascular health in humans. Furthermore, we use a PSM case–control study to verify the positive association between Hcy and CMM found in cross-sectional study, which can enhance the objectivity of our results, using similar covariate distributions to construct case and control groups without affecting the results of the study ( 12 ). As a semi-parametric method, PSM was an important set of tools for estimating causes of disease or treatment effects in observational studies, enabling adjustment for measured confounders in an easy-to-understand and transparent way ( 12 ). However, there were still several potential limitations that should be considered when interpretating the results. Firstly, the plasma Hcy level was only assessed by at only one-time point, which could not successfully reflect the true dynamic levels of individual Hcy exposure. Future studies with repeated assessment are very important and indispensable. Besides, participants in current study are from Hunan province, China, and the sample of this special population is considered large, however, the generalizability of our results may be constrained in other regions in China and in other countries. Therefore, multiprovince or multinational monitoring studies will be necessary. Additionally, some variables, like lifestyle factors, were self-reported and only evaluated at a point in time, and it was not possible to completely avoid recall or evaluation biases. Moreover, due to the cross-sectional and case–control design of this study, causality cannot be inferred. Therefore, further larger prospective investigations are required to confirm our results. Finally, the associations between Hcy and single CMDs (hypertension, diabetes and CHD) are well-established, however, a limited number of studies examined the associations between Hcy and CMM. Our study provides important and new evidence to this fields, however, it still needs to be validated in other additional studies in the future. Section title: Discussion Educational score: 4.027373790740967 Domain: biomedical Document type: Study Language: en In conclusion, our findings add new evidence to this field that of high level of plasma Hcy is consistently associated with higher risk of CMM among Chinses adults, with the largest effect combination of coexistence of diabetes, hypertension and coronary heart disease. These findings have implications for cardiologists that CMM can be attributable to high level of plasma Hcy, and for decision makers that Hcy has become a public threat that persistently affects cardiovascular health in humans. There is an urgent need for a targeted approach to dynamically monitor and reduce plasma Hcy levels to reduce the risk of CMM. | Review | biomedical | en | 0.999999 |
PMC11695424 | Section title: Introduction Educational score: 4.130984306335449 Domain: biomedical Document type: Review Language: en Prostate cancer (PCa) is the second most common cancer worldwide and one of the leading causes of cancer-related death in men, according to the 2020 global cancer data released by the International Agency for Research on Cancer ( 1 ). Although most PCa patients are diagnosed with indolent or slow-progressing disease, approximately 15% of patients are diagnosed with high-risk cancer that can be life-threatening ( 2 ). Additionally, there are significant differences in the incidence and mortality rates of PCa among different racial and ethnic groups. The incidence rate among black patients is 70% higher than that among white patients, and the mortality rate is 2 to 4 times higher compared to other racial and ethnic groups ( 3 ). The treatment of PCa remains a challenging issue for clinicians and researchers, particularly in the case of metastatic disease. Bone metastasis is the most common site of metastasis in PCa and is a major cause of patient mortality. Approximately 1.7% to 11.9% of patients have bone metastasis at the time of initial diagnosis, with a median survival of less than 3 years and a 5-year survival rate of only 3%, severely impacting patients’ quality of life ( 4 , 5 ). Section title: Introduction Educational score: 4.085402011871338 Domain: biomedical Document type: Study Language: en Androgen deprivation therapy (ADT) is the mainstay of treatment for PCa, but although tumors often initially respond to ADT, the treatment effect is usually temporary and frequently leads to the development of resistance ( 6 , 7 ). Data indicate that 10% to 20% of PCa progresses to castration-resistant prostate cancer (CRPC) within 5 years of diagnosis, with 84% of newly diagnosed CRPC patients experiencing metastasis ( 8 ). The median survival period for patients after the diagnosis of CRPC is approximately 14 months, which contributes to the increased mortality rate of PCa ( 7 ). Section title: Introduction Educational score: 3.935748338699341 Domain: biomedical Document type: Study Language: en For patients with locally advanced high-grade disease who are not eligible for radical prostatectomy, external beam radiation therapy (EBRT) is commonly employed as the primary treatment modality ( 9 ). Although it is known that the addition of ADT to EBRT improves overall survival (OS) in patients with Gleason scores of 8-10 by approximately 1.5-fold compared to ADT alone ( 10 , 11 ), the survival benefit from adding ADT is less pronounced for Gleason scores of 9-10, suggesting a lower sensitivity to ADT ( 12 ). Section title: Introduction Educational score: 4.219529151916504 Domain: biomedical Document type: Review Language: en Currently, non-hormonal treatment options for advanced PCa mainly include chemotherapy and immunotherapy. Although studies have shown that the use of chemotherapy drugs may be beneficial in terms of survival, such as the efficacy of Docetaxel in symptomatic or rapidly progressing CRPC ( 13 ), the cytotoxic potential diminishes as androgen receptor-targeted therapy becomes the frontline treatment for resistant PCa. The rapid development of immunotherapy has brought revolutionary changes to the field of cancer treatment. However, the use of any single immunotherapy modality is unlikely to significantly alter the outcomes of PCa. Studies have shown that combining cancer vaccines or immune checkpoint inhibitors with other immunotherapeutic agents, hormonal therapies, radiation therapy, DNA damaging agents, or chemotherapy can enhance immune functionality and provide clinical benefits ( 14 , 15 ). However, the limitations of immunotherapy are also quite apparent, including limited efficacy in the majority of patients, low response rates, and the potential for immune-related adverse effects, which are significant contributors to treatment failure in PCa. Furthermore, current treatment approaches have not fully addressed individual variability, resulting in a lack of broad applicability. Future research should focus on exploring novel immunotherapeutic strategies, optimizing combination therapies, and enhancing the study of patient biomarkers to improve efficacy and ensure a wider spectrum of beneficiaries ( 16 ). Section title: Introduction Educational score: 4.241008758544922 Domain: biomedical Document type: Study Language: en PCa is a disease characterized by significant heterogeneity, and the mechanisms underlying its occurrence and progression are complex and dynamic. Previous research has highlighted the interaction between malignant epithelial cells and the tumor microenvironment (TME) as a key driver of PCa progression ( 17 ). Furthermore, we believe that cellular heterogeneity plays a critical role within the TME of PCa. Cellular heterogeneity reflects the diversity among different cell populations in the PCa microenvironment, which not only directly influences tumor growth and dissemination mechanisms but also establishes complex interactions among these cells. Investigating this heterogeneity can help elucidate the evolutionary pathways of PCa and deepen our understanding of its progression mechanisms, thereby providing essential insights for developing more targeted therapeutic strategies. In recent years, the development of single-cell sequencing technology has provided a new tool for studying the heterogeneity of tumor cells and the TME. By evaluating thousands of cells simultaneously, single-cell sequencing technology can reveal the complexity of intratumoral cells and provide new insights into the field of tumor biology, thereby improving the diagnosis and treatment of PCa and enhancing patient prognosis and survival rates. Therefore, we performed single-cell sequencing analysis on a PCa dataset from the GEO database, providing new perspectives for the diagnosis and treatment of PCa to improve patient prognosis and survival rates. Section title: Acquisition and processing of single-cell derived data Educational score: 3.985121011734009 Domain: biomedical Document type: Study Language: en The PCa data obtained through scRNA-seq was retrieved from the NCBI Gene Expression Omnibus (GEO) database ( https://www.ncbi.nlm.nih.gov/geo/ ). The dataset used for single-cell analysis included 32 tumor and non-tumor samples from 18 PCa patients, with the GSE accession number GSE181294. Detailed information about the samples, including Tumor, Adj-normal, grade, Gleason, Grade Group, Path Stage, Margin, Age, etc., can be found in Supplementary Table S1 . Bulk RNA-seq datasets and clinical data were obtained from the Cancer Genome Atlas (TCGA) ( https://portal.gdc.cancer.gov/ ). The data used in this study was obtained from publicly available databases and therefore did not undergo ethical review. Section title: Acquisition and processing of single-cell derived data Educational score: 3.9736738204956055 Domain: biomedical Document type: Study Language: en The scRNA-seq data was imported into R software (version 4.2.0) and examined using the Seurat package (version 4.3.0) ( 18 – 21 ). We performed rigorous quality control on the data excluding the following cells: (1) 300 nFeature < 7,500; (2) 500 nCount < 100,000; (3) mitochondrial gene expression exceeding 25% of the total gene count within the cell; (4) red blood cell gene expression exceeding 5% of the total gene count within the cell. We kept 127,930 cells ultimately for more study. Our research did not call for ethical permission as we made use of publicly accessible database data. Section title: Acquisition and processing of single-cell derived data Educational score: 4.1023335456848145 Domain: biomedical Document type: Study Language: en Normalization and selection of the top 2000 highly variable genes ( 22 – 26 ) were performed on the filtered samples using the “NormalizeData” and “FindVariableFeatures” functions in the Seurat package. To correct for batch effects between datasets, principal component analysis was conducted using the Harmony R package (version 0.1.1) ( 27 , 28 ). Cells were clustered using the FindClusters function with a resolution of 1.0 based on the top 30 principal components (PCs). The top 30 significant PCs were selected for uniform manifold approximation and projection (UMAP) dimensionality reduction and visualization of gene expression ( 29 , 30 ). Section title: Single cells copy number variation evaluation Educational score: 4.060494422912598 Domain: biomedical Document type: Study Language: en The scRNA-seq data was analyzed for CNVs using the inferCNV R program (version 1.6.0) available from the GitHub repository of the Broad Institute ( https://github.com/broadinstitute/inferCNV ). This software application facilitates the differentiation between malignant and healthy cells by examining the chromosomal positions and gene expression levels to ascertain CNVs ( 31 , 32 ). Tumor-EPCs were identified as cells with high CNV scores. Section title: Cell type identification Educational score: 4.055482387542725 Domain: biomedical Document type: Study Language: en We used Seurat’s “FindAllMarkers” function ( 33 ) to conduct a Wilcoxon rank-sum test with the goal of identifying differentially expressed genes (DEGs) among various cell clusters in order to examine the heterogeneity of PCa cells ( 34 ). Threshold = 0.25, min.pct = 0.25, and min.diff.pct = 0.25 were the parameters that were employed. Then, we used Seurat’s “DotPlot” and “featureplot” programs to show the expression patterns of the DEGs in each cluster. Manually reviewing the outcomes and consulting pertinent literature helped with the cell annotation process. Additionally, we regrouped these cells in order to investigate the heterogeneity of malignant cells in greater detail. We used marker identification to characterize each subtype based on the genes unique to that grouping. Section title: Enrichment analysis Educational score: 4.034121513366699 Domain: biomedical Document type: Study Language: en The Gene Ontology (GO) ( 35 , 36 ) and Gene Set Enrichment Analysis (GSEA) ( 37 ) tools were used to conduct enrichment studies of DEGs in distinct cell types. We performed a functional analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) utilizing the ClusterProfiler R package (version 4.6.2) ( 38 – 42 ). Section title: Enrichment analysis Educational score: 3.8201944828033447 Domain: biomedical Document type: Other Language: en One technique for gene set enrichment analysis is gene set variation analysis (GSVA). In order to determine enrichment scores for each gene set in each sample, it evaluates the variability of gene expression data and compares it to predefined gene sets. Section title: Pseudotime analysis and cell fate analysis Educational score: 4.081532955169678 Domain: biomedical Document type: Study Language: en In order to examine the variations in development and differentiation amongst malignant subtypes in PCa, we created pseudotime trajectories of the subtypes using Monocle (v2.24.0), which showed the patterns of malignant cell differentiation ( 43 ). We utilized the CytoTRACE R package (version 0.3.3) ( 44 ) to evaluate cell fate, which enabled us to deduce the time course of cell differentiation. Next, we employed the Slingshot software (version 2.6.0) to deduce cell lineages as malignant subtypes differentiated ( 45 ). Each malignant subtype’s differentiation trajectory was deduced using the “getlineage” and “getCurves” tools. After that, a UMAP projection was created using these trajectories for visualization. Section title: Cell-cell communication analysis Educational score: 4.039684772491455 Domain: biomedical Document type: Study Language: en We used the CellChat R package (version 1.6.1) ( 46 ) to compute regulatory networks based on ligand-receptor levels and infer complicated cell-to-cell interactions, as well as analyze the intercellular communication network within the TME. The “Identify Communication Patterns” function was used to estimate the number of communication patterns, and the “netVisualDiffInteraction” function was used to show the variation in communication strength between cells. The P -value, or significance threshold of 0.05, was used. Section title: Gene regulatory network construction Educational score: 3.944096326828003 Domain: biomedical Document type: Study Language: en To analyze the scRNA-seq data and reconstruct gene regulatory networks, we used Python (v3.7) and the pySCENIC package (version 0.10.0) uncovering key gene regulatory mechanisms. To evaluate TFs enrichment and regulatory factor activity, we created an AUCell matrix for this investigation. Section title: Construction and validation of risk model Educational score: 4.194953918457031 Domain: biomedical Document type: Study Language: en To construct a risk model, we obtained PCa-related data from the TCGA database ( https://portal.gdc.cancer.gov/ ). Initially, we performed univariate Cox regression analysis to screen for potential prognostic-related genes ( 47 – 52 ). To account for multicollinearity among these genes, we further employed the least absolute shrinkage and selection operator (LASSO) regression ( 53 – 57 ) using the glmnet package (version 4.1-6). We assigned weights to each gene based on its expression levels and the coefficients obtained from the multivariable Cox regression analysis. This allowed us to construct a risk score formula: Risk score = ∑_i^n (Xi × Yi) (X: coefficient, Y: gene expression level). Next, using the “surv_cutpoint” function, we determined the optimal cutoff value to divide patients into high and low-risk groups based on the risk scores. The predictive accuracy of the model was assessed using ROC curve analysis ( 58 ). To observe the prognosis of patients in different groups, Kaplan-Meier survival curves were used to evaluate the survival differences between different risk groups ( 58 , 59 ). Survival analysis was performed using the Survive package (version 3.3.1) and survminer package (version 0.4.9). To validate the predictive capabilities of the model based on NEFH + malignant cell scores, we employed the “Survival” and “Time ROC” R packages to generate ROC curves for 1-year, 3-year, and 5-year survival rates, and calculated the area under curve (AUC) value of the ROC curve ( 60 – 65 ). Survival analysis and time-dependent ROC analysis were used for model validation. The distribution of risk score scores, scatter plots of survival status, and heatmaps were utilized to assess the model. Section title: TME immunoassay Educational score: 4.124194622039795 Domain: biomedical Document type: Study Language: en For the evaluation of the TME, we employed the ESTIMATE R package (version 1.0.13) to estimate the stromal score, immune score, and ESTIMATE score in PCa tissues ( 66 ). To analyze RNA-Seq data and determine the relative proportion of infiltrating immune cells, we utilized the Cell Type Identification for Estimating Relative Subtypes of RNA Transcripts (CIBERSORT R package, version 0.1.0) algorithm ( 67 , 68 ), which provides insights into 22 different immune cell types. Additionally, to quantify immune cell infiltration in each sample, we employed the xCell package to assess the enrichment of immune cells in PCa samples. Furthermore, we investigated the correlation between risk scores and immunomodulatory genes, particularly immune checkpoints. To evaluate the response to tumor immune therapy, we utilized the Tumor Immune Dysfunction and Exclusion (TIDE) tool ( http://tide.dfci.harvard.edu ). Section title: Mutation analysis Educational score: 4.0362019538879395 Domain: biomedical Document type: Study Language: en Using the TCGAbiollinks and maftools R package, the somatic mutation data of PCa were obtained from the TCGA database. The PCa expression data and TMB file were imported into the R package. The correlation between DEGs associated with PCa and tumor mutation burden was computed. Based on the correlation results, waterfall plots were generated to depict the high-risk and low-risk groups. Section title: Drug sensitivity analysis Educational score: 4.080424785614014 Domain: biomedical Document type: Study Language: en With the pRRophetic R package (version 0.5), we employed the GDSC database ( https://www.cancerrxgene.org/ ), the most comprehensive pharmacogenomics database, to predict the treatment response of each tumor sample ( 69 , 70 ). The IC50 values for each medication were determined using regression, and the accuracy of both regression and prediction was assessed using 10-fold cross-validation with the GDSC training set. All settings were set to their default values, including the “combat” option for removing batch effects and the mean value for duplication gene expression ( 71 – 73 ). Section title: Cell culture Educational score: 4.087231159210205 Domain: biomedical Document type: Study Language: en The MDA PCa 2b and VCap cell lines were acquired from ATCC and cultured in specific growth media. MDA PCa 2b cells were cultured in F12K medium, while VCap cells were cultured in DMEM medium. Both media were supplemented with 10% fetal bovine serum from Gibco BRL, USA, and 1% penicillin/streptomycin. The cells were incubated under standardized conditions, including a constant temperature of 37°C, a carbon dioxide concentration of 5%, and a humidity level of 95%. Section title: SiRNA transfection Educational score: 4.086427688598633 Domain: biomedical Document type: Study Language: en IRX4 knockdown was achieved by employing short interfering RNA constructs obtained. The transfection process followed the prescribed guidelines supplied by Lipofectamine 3000 RNAiMAX (Invitrogen, USA). The cells were introduced to a six-well plate when they reached a 50% coverage and subsequently underwent transfection using negative controls (si-NC) as well as knockdown constructs (Si-IRX4-1 and Si-IRX4-2). The transfection process utilized Lipofectamine 3000 RNAiMAX (Invitrogen, USA) for each instance. The siRNA sequences are recorded in Supplementary Table S2 in the Supplementary Materials . Section title: Cell viability assay Educational score: 4.096676826477051 Domain: biomedical Document type: Study Language: en The CCK-8 assay was used to evaluate the cellular vitality of MDA PCa 2b and VCap cells after transfection. The cell suspensions were added to a 96-well plate with a density of 5×10³ cells per well and incubated for 24 hours. After adding 10μL of CCK-8 labeling reagent (A311-01, Vazyme) to each well, the plate was placed in a light-protected environment and incubated at 37°C for 2 hours. Cell viability was assessed by quantifying the absorbance at 450nm using an enzyme-linked immunosorbent assay (ELISA) reader on days 1, 2, 3, and 4. The mean optical density (OD) values were computed and depicted on a line graph. Section title: Cell proliferation assay with 5-Ethynyl-2’-deoxyuridine Educational score: 4.1675214767456055 Domain: biomedical Document type: Study Language: en MDA PCa 2b and VCap cells that had been genetically modified were placed in a 6-well plate at a concentration of 5×10³ cells per well and let to grow overnight. Afterwards, a solution of EdU with a concentration that is twice as strong as the original was prepared by combining 10 mM EdU with a medium that does not contain serum. The solution was introduced into the cell culture and permitted to incubate at a temperature of 37°C for a duration of 2 hours. After the incubation period, the liquid containing the cells was removed, and the cells were carefully rinsed with PBS. Subsequently, the cells were treated with a 4% paraformaldehyde solution for a duration of 30 minutes to ensure fixation. Afterwards, a glycine solution with a concentration of 2 mg/mL and 0.5% Triton X-100 was administered for a duration of 15 minutes. Subsequently, the cells were incubated at room temperature with a solution containing 1 ml of 1X Apollo and 1 ml of 1X Hoechst 33342 for a duration of 30 minutes. Fluorescence microscopy was used to measure and analyze cell proliferation. Section title: Wound healing assay Educational score: 4.125958442687988 Domain: biomedical Document type: Study Language: en The cells were transfected, then grown to a cell density of 95% by seeding them onto 6-well plates. Then, using a sterile 200 μL pipette tip, a careful linear scratch was made over the cell layer in the culture wells. After that, PBS was used to gently wash the wells. After rinsing, the cell culture was let to continue and the culture medium was changed. Photography was used to record the scratches at the original time point (0 hours) and 48 hours later. Measurements were made of the scratches’ width for further examination. Section title: Transwell assay Educational score: 4.11435079574585 Domain: biomedical Document type: Study Language: en To prepare for the experiment, the cells were subjected to a 24-hour serum-free medium starvation. Subsequently, cell suspensions were added to the upper chamber, which contained Costar, after the addition of matrix gel (BD Biosciences, USA). In the lower chamber, serum-containing medium was added. The cells were then incubated in a cell culture incubator for 48 hours. After the incubation period, the cells were fixed with 4% paraformaldehyde and stained with crystal violet to evaluate their invasive capacity. Section title: Statistical analysis Educational score: 2.9890644550323486 Domain: biomedical Document type: Study Language: en We performed statistical analysis using R software and Python software to analyze the database data. All p -values reported in this study are two-tailed, with values less than 0.05 considered statistically significant. P -values below 0.001 were considered highly significant, while those below 0.0001 were regarded as extremely significant. Section title: Single cell landscape of PCa Educational score: 4.1125335693359375 Domain: biomedical Document type: Study Language: en We conducted an extensive analysis of the obtained dataset to unveil the intricate single-cell landscape within the PCa microenvironment. Our workflow was illustrated in Figure 1 . Using the known typical cell type marker genes, 127,930 high-quality cells were labeled. By using dimensionality reduction clustering with UMAP plot, 9 cell types were obtained: T-NK cells, B-plasma cells, endothelial cells (ECs), myeloid-cells, mast cells (MCs), epithelial cells (EPCs), pericytes-smooth muscle cells (pericytes-SMCs), fibroblasts, and plasmacytoid dendritic cells (pDCs). The proportions of different tissue types (Adjacent-Normal high-grade (NHG), Adjacent-Normal low-grade (NLG), Tumor high-grade (THG, Gleason 8–10), Tumor low-grade (TLG, Gleason 6 and 7) and cell cycle phases (G1, G2/M, S) in various cell types were visualized using pie charts . Section title: Single cell landscape of PCa Educational score: 4.141430377960205 Domain: biomedical Document type: Study Language: en Subsequently, we analyzed the proportion of cell types in different phases . We found that EPCs, pericytes-SMCs, fibroblasts, ECs, and myeloid-cells were mainly derived from tumor cells. The results of cell cycle study showed that T-NK cells occupied a larger proportion in G2/M and S phases. Then, we described the expression level and distribution of typical marker genes related to cell subtypes in cells . By visualizing the analysis of G2/M.score, S.score, nFeature-RNA and nCount-RNA of all cells, the differences among cell types were further clarified . The results of enrichment analysis showed that the marker genes of EPCs were mainly enriched in oxidative phosphorylation pathway . It is worth noting that PCa is mainly transformed from glandular epithelial cells of prostate ( 74 ), and oxidative phosphorylation is considered as a tumor-related metabolic marker ( 75 ), which confirms that our findings are consistent with the recognized biological functions related to PCa. Section title: Visualization of malignant cell subtypes in PCa Educational score: 4.293604373931885 Domain: biomedical Document type: Study Language: en Given the profound importance of malignant cells in TME, our subsequent objective is to characterize these cells in the microenvironment of PCa. Studies indicated that a high CNV score typically signified significant copy number alterations within cells, which might have been associated with malignant transformation. CNV scoring could be utilized to identify tumor heterogeneity and reveal the presence of different subpopulations within the tumor microenvironment, aiding in the understanding of tumor development and resistance mechanisms ( 76 , 77 ). To detect aberrant amplification or deletion of chromosome copy number in EPCs, we initially employed inferCNV to analyze the chromosome CNV of epithelial cells using endothelial cells as a reference . According to CNV level, malignant cells was distinguished from EPCs. After that, we re-clustered 3,076 malignant cells, and annotated them according to each cell marker gene, and identified four malignant cell subtypes: C0 TRPM4 + malignant cells, C1 SLPI + malignant cells, C2 MIR205HG + malignant cells and C3 NEFH + malignant cells . We employed the UMAP plot combined with a cellular proportion pie chart to illustrate the relative distribution of different subgroups (THG and TLG) within the four malignant cell subtypes . Additionally, we visualized these cells based on tissue types . The results demonstrated that, compared to other subtypes, the C1 and C3 malignant cell subtypes exhibited a higher proportion of THG tissue. Additionally, a stacked bar graph revealed that THG tissue predominantly comprised the C1 and C3 subtypes, in contrast to TLG tissue . Therefore, we hypothesized that the heterogeneity between these two tissue types may be associated with the C1 and C3 subtypes. Similarly, the Ro/e preference plot indicated a higher cell abundance of C1 and C3 subtypes in THG tissue, further substantiating our conclusion . Figure 3G showcased the differential expression of the top five marker genes in the malignant cell subtypes, visualized using the volcano plots . Section title: Visualization of malignant cell subtypes in PCa Educational score: 4.0777788162231445 Domain: biomedical Document type: Study Language: en Next, we showed the results of CNVscore, nFeature-RNA and nCount-RNA of different tissue types and malignant cell subtypes by violin plots . It was found that the CNVscore of THG was higher than that of TLH, which was consistent with the biological process of PCa tissue. It is noteworthy that both the C1 and C3 subtypes exhibited higher CNV scores. Additionally, compared to the C1 subtype, the C3 subtype with high NEFH expression showed elevated levels of nCount-RNA and nFeature-RNA. Therefore, we inferred that the C3 subtype’s malignancy level might have been higher. Section title: Visualization of malignant cell subtypes in PCa Educational score: 4.106010437011719 Domain: biomedical Document type: Study Language: en Subsequently, we determined that the subtypes shared specific biological functions through the enrichment analysis of a variety of malignant cells. For instance, transition metal homeostasis was associated with both C1 and C3 subtypes. Furthermore, the C3 subtype was enriched in the cholesterol metabolism process . We also illustrated the metabolism pathways using bubble plot and discovered that the C3 subtype was substantially enriched in the metabolic pathways of glutathione metabolism and fatty acid biosynthesis . Section title: Unveiling the development and differentiation characteristics of malignant cell subtypes through pseudotime analysis Educational score: 4.234808921813965 Domain: biomedical Document type: Study Language: en To understand the source and development of cancer cells, we extensively studied the intricate lineage and advancement of malignant cells utilizing the Monocle software. It was easy to see in Figures 4A–E that the C2 MIR205HG+ malignant cells were mostly in the early stages of differentiation, more specifically in state 1 along the time axis. Conversely, the C3 NEFH+ malignant cells were in the last stage of development, predominantly found within state 3 throughout the temporal trajectory. Afterwards, we utilized the CytoTRACE technique to evaluate the differentiation and developmental correlation between several subtypes of tumor cells. The findings indicated that C3 NEFH+ malignant cells displayed elevated cellular stemness, as seen in Figure 4F . Combining the Slingshot analysis, we discovered a differentiation boundary transitioning from TLG to THG tissue types, indicating a higher malignant degree among the cells at the terminal stage of differentiation . Malignant cells often possess self-renewal capability and differentiation potential. Thus, as the tumor advances, malignant cells in the last stage of differentiation tend to have greater cellular stemness, in line with the results obtained from the CytoTRACE investigation. Furthermore, the progression of cancerous cell subtypes can be described in the order of C2→C1→C0→C3. Additionally, the slingshot analysis of time states showed that State 3 was positioned at the end of one of the differentiation branches, with a greater proportion of C3 subtypes within state 3. This finding further supports the conclusions drawn from the Monocle analysis . Section title: Unveiling the development and differentiation characteristics of malignant cell subtypes through pseudotime analysis Educational score: 4.117402076721191 Domain: biomedical Document type: Study Language: en Consistent with those findings, the genes TRPM4 , which serve as markers for the C0 subtype, and NEFH , which serve as markers for the C3 subtype, were predominantly expressed during the mid-late stage of the developmental trajectory. On the other hand, the expression levels of SLPI , a marker for the C1 subtype, and MIR205HG+ , a marker for the C2 subtype, were initially high but declined over time . In addition, we conducted GO-BP enrichment analysis on the DEGs linked to the malignant cell subtypes. The analysis revealed that these genes were mainly enriched in biological processes related to immunity, cytotoxicity, antigen, processing, and other similar activities . Section title: Cell-cell communication and visualization of the PTN signaling pathway Educational score: 4.094972610473633 Domain: biomedical Document type: Study Language: en We utilized CellChat to infer and analyze communication between tumor cell subtypes and other cell types from single-cell data ( Supplementary Table S3 ). The number and intensity of interactions between all cell types in PCa samples were comprehensively summarized . It was found that compared with other types of cells, C3 NEFH+ malignant cells had a more significant effect on pericytes-SMCs and fibroblasts. The circle graphs quantified the number and intensity of interactions between all cells with C3 NEFH+ malignant cells as the signal source and fibroblasts as the target respectively . The results showed that there was a strong intercellular communication network between C3 NEFH+ malignant cells and fibroblasts. Section title: Cell-cell communication and visualization of the PTN signaling pathway Educational score: 4.096198081970215 Domain: biomedical Document type: Study Language: en Next, we identified the ligand-receptor signals associated with the communication pathway to determine the primary afferent and efferent signals related to the C3 NEFH+ malignant cell subtype and other cells. The findings indicated that the primary ligands associated with the output of C3 NEFH+ malignant cells, when employed as signal senders, were MIF, MK, GDF, and CD46. As signal receivers, the fibroblast-related receptors mainly included PTN, CD99, and PDGF. Section title: Cell-cell communication and visualization of the PTN signaling pathway Educational score: 4.272430896759033 Domain: biomedical Document type: Study Language: en Subsequent analysis revealed potential connections to the PTN signaling pathway network. Through network centrality analysis of the inferred PTN signaling network, we found that C3 NEFH+ malignant cells can act as signal senders within the PTN pathway. Fibroblasts, on the other hand, can function both as signal sender promoting their transformation into cancer-associated fibroblasts (CAFs) and as signal receiver, mediator, and influencer interacting with C3 NEFH+ malignant cells . Notably, C3 NEFH+ malignant cells demonstrated the ability to engage in paracrine interactions with fibroblasts, resulting in a substantial communication intensity between these cell populations . In addition, we compared the receptor-ligand interaction between C3 NEFH+ malignant cells and other cell types and found that when this subtype interacted with fibroblasts, the ligand receptor had a high communication probability with PTN-NCL . Additionally, a circle graph further confirmed that the interactions between C3 NEFH+ malignant cells and fibroblasts could be mediated through the receptor-ligand pairs within the PTN signaling pathway, specifically involving PTN-NCL . Section title: Cell-cell communication and visualization of the PTN signaling pathway Educational score: 4.046377182006836 Domain: biomedical Document type: Study Language: en Essentially, our study provided profound insights into the intricate interactions between fibroblasts and malignant cell subtypes in PCa. This relationship is likely closely linked to the transformation of fibroblasts into CAFs, which promotes the progression of PCa. Section title: Identification and analysis of TFs regulatory modules Educational score: 3.350867509841919 Domain: biomedical Document type: Other Language: en TFs can directly interact with the genome and regulate gene transcription by binding to specific nucleotide sequences upstream of the target gene. This interaction plays a significant role in determining the biological functions of cells. Section title: Identification and analysis of TFs regulatory modules Educational score: 3.9215877056121826 Domain: biomedical Document type: Study Language: en To begin with, we employed the SCENIC and connection specificity index matrix to classify prostate malignant cells into four regulatory modules (M1, M2, M3, M4) based on the similarity of AUCell score rules . Subsequently, we conducted dimensionality reduction and clustering analyses considering various subtypes and tissue types . Section title: Identification and analysis of TFs regulatory modules Educational score: 4.140603542327881 Domain: biomedical Document type: Study Language: en Through a comparison of the expression levels and regulatory activities of TFs within each module and the malignant cell subtypes, we identified that the TFs in the M4 module predominantly regulated C3 NEFH+ malignant cells . Within the M4 module, we found that IRX4 exhibited the highest fraction of variance across the subtypes, indicating its prominent role in explaining a significant portion of the data variability within the M4 module . Importantly, IRX4 demonstrated a high specificity score in C3 NEFH+ malignant cells and THG . This suggested a strong and specific regulatory relationship between IRX4 and its target genes, highlighting its potential as a biomarker or therapeutic target. Section title: Identification and analysis of TFs regulatory modules Educational score: 4.051203727722168 Domain: biomedical Document type: Study Language: en Finally, we visualized the expression levels of five key regulatory factors (NEUROD1, IRX4, LTF, PURA, and ELK4) in the C3 subtype . We observed that the expression of IRX4 in the C3 subtype was significantly higher compared to other malignant cell subtypes. To further investigate the relevance of these TFs, we performed survival analysis using Kaplan-Meier and AUC curves . Interestingly, our results indicated that high expression of IRX4 might be associated with a poorer prognosis in PCa. Nevertheless, the specific mechanism by which IRX4 influences PCa remains unclear. Therefore, conducting in vitro functional experiments to validate the impact of IRX4 on PCa cells is imperative. Section title: In vitro experimental verification Educational score: 4.039583206176758 Domain: biomedical Document type: Study Language: en To further investigate the role of IRX4 in PCa, we conducted in vitro experiments using MDA PCA 2b and VCap cell lines. Initially, we knocked down IRX4 and assessed the mRNA and protein expression levels before and after knockdown. Our findings revealed a significant decrease in mRNA and protein expression levels in both cell lines compared to the control group . Moreover, there was a noticeable decrease in cell viability following the knockdown . Section title: In vitro experimental verification Educational score: 4.110969543457031 Domain: biomedical Document type: Study Language: en Subsequent colony experiments demonstrated a significant reduction in the number of cells after IRX4 knockdown . Additional EDU experiment confirmed that the knockout of IRX4 partially inhibited cell proliferation . Furthermore, the wound healing assay and Transwell assay indicated a substantial decrease in cell migration after IRX4 knockdown , and the cell invasion capability also decreased . Section title: In vitro experimental verification Educational score: 4.022472858428955 Domain: biomedical Document type: Study Language: en Collectively, these results indicate that the knockdown of IRX4 can inhibit the activity, migration, and proliferation of tumor cells, thereby impeding tumor growth. Section title: Construction and correlation analysis of risk prediction model Educational score: 4.1093902587890625 Domain: biomedical Document type: Study Language: en We developed a prognostic model to investigate the clinical significance of the NEFH+/IRX4 regulatory network. Initially, we performed univariate Cox regression analysis to identify genes significantly associated with prognosis . To address the issue of multicollinearity among these genes, we employed LASSO regression analysis for further selection . Subsequently, a multivariate Cox regression analysis was conducted, resulting in the identification of five genes related to prognosis. The coef values for these genes were calculated . The findings revealed that ZNF782, ZNF695, YY1, NR1I3 , and FOXA3 were unfavorable prognostic factors. Section title: Construction and correlation analysis of risk prediction model Educational score: 4.1549272537231445 Domain: biomedical Document type: Study Language: en To further investigate the differences between different scoring groups, we performed an analysis of DEGs. Based on the optimal cut-off value of NEFH+ malignant cell score, patients in the TCGA cohort were categorized into two groups: the high NmRS group and the low NmRS group (NmRS: NEFH+ malignant cells risk score). It was observed that higher scores were associated with worse prognosis. Curve and scatter plots were utilized to illustrate the differences in risk scores, survival, and outcomes between the two groups, clearly indicating that the high NmRS group was associated with a poorer prognosis . Furthermore, a heatmap was generated to display the differential expression of the five genes between the high and low NmRS groups . Principal component analysis demonstrated that PC1 (high NmRS group) accounted for 10.2% of the total variance in all principal components, while PC2 (low NmRS group) accounted for 4.2% of the total variance . Additionally, the ROC curve provided an intuitive visualization of the AUC values predicted by the TCGA cohort at 1 year, 3 years, and 5 years, demonstrating the predictive value of the model . The Kaplan-Meier survival curve further confirmed the conclusion that the high NmRS group had a worse survival outcome, with a p-value less than 0.0001 . Section title: Construction and correlation analysis of risk prediction model Educational score: 3.8447773456573486 Domain: biomedical Document type: Study Language: en To elucidate differential gene expression and associated biological processes between high and low groups, we employed visualization and enrichment analysis techniques. Initially, a heatmap was utilized to depict gene expression of the top thirty genes , while a volcano plot showcased differential gene up-regulation and down-regulation . Section title: Construction and correlation analysis of risk prediction model Educational score: 4.156883239746094 Domain: biomedical Document type: Study Language: en Subsequently, various enrichment methods were employed to delve deeper into the related biological processes. KEGG analysis indicated predominant enrichment of differential genes in cytoskeleton in muscle cells, motor proteins, and cardiac muscle contraction . Conversely, GO analysis revealed enrichment in myofibril and muscle cell development . Furthermore, GSVA enrichment analysis was conducted on the gene set comprising the prediction model, as represented in the heatmap . Lastly, GSEA analysis was performed on the initial 30 up-regulated and down-regulated genes. Up-regulated genes exhibited enrichment in DNA geometric change, sister chromatid segregation, mitotic sister chromatid segregation, cell cycle DNA replication, down-regulated genes were primarily enriched in striated muscle adaptation, sarcomere organization, striated muscle cell development, and muscle cell development . Section title: Analysis of immune infiltration, mutation, and drug sensitivity Educational score: 4.119629383087158 Domain: biomedical Document type: Study Language: en To investigate immune cell composition differences in NmRS with varying risk scores, we employed the CIBERSORT algorithm for analyzing twenty-two immune cell types in TCGA database PCa patients, as illustrated in Figure 10A . The proportions of eight immune cell categories were presented in Figure 10B , revealing distinctions between the two groups via a box plot. Notably, higher infiltration of naive B cells, T cells CD4 memory resting, and T cells follicular helper was observed in the high NmRS group, implying potential immune reactions associated with immune escape. Section title: Analysis of immune infiltration, mutation, and drug sensitivity Educational score: 4.064581394195557 Domain: biomedical Document type: Study Language: en Subsequently, we assessed the correlation between immune cells and NmRS, as depicted in Figure 10C . The findings indicated a significant positive correlation between NmRS and macrophages, T cells CD4 memory resting, and T cells follicular helper, while a significant negative correlation was observed between NmRS and M1 macrophages. A heatmap visualization was employed to depict the correlation analysis among immune cells, modeling genes, OS, and risk score . Moreover, differences in TIDE values between the two groups were evident . Section title: Analysis of immune infiltration, mutation, and drug sensitivity Educational score: 4.025506019592285 Domain: biomedical Document type: Study Language: en Additionally, Figure 10F displayed a heatmap showcasing variations in modeling genes, StromalScore, ImmuneScore, ESTIMATEScore, TumorPurity, and immune cell infiltration levels between the high NmRS and low NmRS groups, computed using the CIBERSORT and Xcell algorithms. Section title: Analysis of immune infiltration, mutation, and drug sensitivity Educational score: 4.026183605194092 Domain: biomedical Document type: Study Language: en Next, we presented the correlation between DEGs and tumor mutation load using a waterfall diagram for the high and low-risk groups . Furthermore, the correlation between immune checkpoint-related genes, modeling genes, risk score, and OS was displayed in a bubble plot . The results revealed a strong positive correlation between NR1I3, ZNF782, YY1 , and most immune checkpoints. Section title: Analysis of immune infiltration, mutation, and drug sensitivity Educational score: 4.027987480163574 Domain: biomedical Document type: Study Language: en Finally, through drug sensitivity analysis, we identified potential clinical efficacy of certain drugs for prognosis-related genes, including ABT.263, AZD.2281, Bleomycin, Cisplatin, Cytarabine, Embelin, Epothilone.B, Etoposide, IPA.3, Methotrexate, MS.275, Shikonin, and Vorinostat. However, for the low-risk group, Dasatinib and Bicalutamide were found to be effective . Section title: Discussion Educational score: 4.4890289306640625 Domain: biomedical Document type: Study Language: en PCa is a prevalent malignancy of the male reproductive system, and its treatment continues to face numerous challenges, particularly in the management of metastatic disease. Treatment resistance is one of the primary factors leading to therapeutic failure in PCa, significantly impacting patient prognosis ( 78 ). Research has shown that high-grade PCa with Gleason scores of 8-10 exhibit rapid growth, increased metastatic risk, and heightened resistance to treatment ( 79 ). High-grade PCa exhibited more aggressive biological behavior, often characterized by faster growth and an earlier tendency to metastasize. The rapid progression of these tumors could have been linked to the genetic instability of tumor cells, the activation of the EMT process, and the inflammatory responses within the tumor microenvironment ( 80 ). The metabolic reprogramming in high-grade PCa likely involved changes in lipid metabolism. Studies indicated that prostate cancer cells were more dependent on fatty acid oxidation as the primary energy supply pathway, with relatively lower glucose uptake rates ( 81 ). To address these challenges, the rapid development of scRNA-seq in recent years has provided powerful tools for cancer immunology research. These technologies facilitate an in-depth analysis of cellular interactions within the TME and their roles in disease progression, thus offering new perspectives for exploring treatment strategies ( 82 ). Section title: Discussion Educational score: 4.20391845703125 Domain: biomedical Document type: Study Language: en To elucidate the complexities of the PCa TME, we employed scRNA-seq to depict the overall landscape of the TME. Dimensionality reduction and clustering analyses revealed a significant increase in the proportion of tumor cells among various stromal cell types, including EPCs, pericyte-SMCs, fibroblasts, ECs, and myeloid-cells. Notably, PCa primarily originates from prostatic glandular epithelial cells. Furthermore, previous studies have indicated that stromal cells, such as fibroblasts and smooth muscle cells, are crucial components of the PCa microenvironment and are closely associated with the malignant transformation of EPCs and cancer progression ( 83 ). Section title: Discussion Educational score: 4.088052272796631 Domain: biomedical Document type: Study Language: en Additionally, inflammatory responses are recognized as a significant hallmark of cancer. In PCa patients, the expansion of myeloid cells in peripheral blood is often correlated with a shortened tumor survival period and resistance to treatment ( 84 ). Studies have indicated that myeloid inflammatory cells play critical roles in promoting the progression of PCa and treatment resistance ( 85 ). This finding aligns with our conclusions and further underscores the interactions among various cellular components in the TME and their impact on PCa progression. Section title: Discussion Educational score: 4.2849836349487305 Domain: biomedical Document type: Study Language: en To reveal the intra-tumor heterogeneity of malignant epithelial cells, we categorized the obtained cells into four subtypes: C0 TRPM4+ malignant cells, C1 SLPI+ malignant cells, C2 MIR205HG+ malignant cells, and C3 NEFH+ malignant cells. TRPM4 was a calcium-activated, non-selective cation channel that was widely expressed in various organs, immune cells, and the central nervous system. It was involved in physiological processes such as circulation, immune response, cancer, and hormone secretion ( 86 ). Secretory leukocyte protease inhibitor (SLPI), a protein with broad anti-inflammatory and immunoregulatory functions, its expression had altered in diabetic nephropathy (DN), likely linked to its role in reducing inflammation and protecting the kidneys from damage ( 87 ). MIR205HG, a long non-coding RNA (lncRNA), played a key role in esophageal cancer, potentially influencing tumor progression by regulating processes such as cell proliferation, migration, invasion, and apoptosis, and it might serve as a potential diagnostic and therapeutic target for esophageal cancer ( 88 ). NEFH (Neurofilament Heavy Chain) is a neurofilament protein primarily found in axons of neurons, where it promoted efficient neural signal transmission and was linked to the diameter of nerve fibers, affecting the speed of signal conduction ( 89 ). Analysis of these subtypes revealed that the C3 NEFH+ malignant cell subtype is closely related to high-grade tumors and exhibits a higher CNV score, indicating greater malignancy. Section title: Discussion Educational score: 4.518037796020508 Domain: biomedical Document type: Study Language: en Understanding the metabolic changes in cancer unveiled critical energy pathways, including glycolysis, oxidative phosphorylation, glutaminolysis, and lipid metabolism, which played essential roles in cancer cells and served as key targets for cancer therapies ( 90 ). Targeting metabolism was also recognized as an important approach to enhance the effectiveness of standard treatments such as chemotherapy, radiotherapy, and immunotherapy. For example, targeting the metabolism of tumor cells could counteract the metabolic adaptations induced by standard therapies, thereby improving their sensitivity ( 91 ). Metabolomics technology was valuable for identifying metabolic reprogramming pathways and key enzymes associated with disease progression and drug resistance, providing insights into their functions and molecular regulatory mechanisms. These findings could identify metabolic vulnerabilities related to disease and resistance, validating their potential as novel molecular targets for new drug development or combination therapies ( 92 ). In addition, our metabolic analysis showed significant enrichment of the C3 subtype in metabolic pathways, such as glutathione metabolism and fatty acid biosynthesis. Tumor cells typically exist in a reductive microenvironment, characterized by elevated levels of glutathione. Previous research has indicated that the baseline glutathione concentration in PCa cells is significantly higher than that in normal cells ( 93 , 94 ). Moreover, excessive dietary fat intake is considered a major risk factor for PCa, with fatty acid metabolism playing an essential role in promoting the proliferation of PCa cells ( 95 ). Therefore, the enrichment of the C3 subtype in these metabolic pathways not only highlights its importance in the metabolic adaptation of PCa cells but may also reflect its potential role in tumor progression and resistance. These findings offer new avenues for metabolic intervention strategies targeting PCa. Section title: Discussion Educational score: 4.606959342956543 Domain: biomedical Document type: Study Language: en Subsequently, through pseudotime analysis, we found that this subtype was located at the terminal end of the developmental trajectory, exhibiting higher stemness characteristics. We observed a differentiation line indicating a transition of tissue types from TLG to THG, suggesting that cells at the differentiation endpoint possessed higher malignancy. Notably, the distribution of the differentiation endpoint closely aligned with that of the C3 subtype. This observation led us to speculate on the intricate relationship between the C3 NEFH+ malignant cell subtype and the progression of high-grade PCa. Moreover, the trajectories we identified provided significant insights into the biological implications of tumor progression. The alignment of the differentiation endpoint with increased malignancy highlighted how the cells’ developmental status could influence their aggressive behavior and overall tumor dynamics. By understanding these trajectories, we could better correlate specific stemness features with the transition to more aggressive cancer phenotypes. This emphasized the critical need to study the C3 subtype as a unique research entity, as it may serve as a key player in the advancement of high-grade PCa and its associated malignancy features. In the TME, the role of the tumor stroma is often perceived as dualistic, with the potential to both inhibit tumor development and promote its progression. In PCa, the mechanisms governing the interactions between epithelial and stromal cells remain insufficiently characterized compared to other malignancies. Fibroblasts, which are crucial components of the stroma, are prevalent in the TME and significantly influence tumor cell proliferation and migration through the secretion of various cytokines, matrix proteins, and growth factors ( 96 ). The TME of PCa was composed of a complex cellular ecosystem that included tumor cells, immune cells, and stromal cells, among other cell types. This heterogeneity was particularly evident in prostate cancer and was closely linked to patient prognosis. While heterogeneity in the TME was also observed in other cancers, such as breast and lung cancer, the cellular makeup and interaction patterns might have differed ( 97 ). In prostate cancer, the infiltration of regulatory T cells (Tregs) was associated with an immunosuppressive microenvironment in advanced disease, possibly induced by a FAP+ fibroblast subpopulation ( 98 ). Moreover, various cell subpopulations and transcriptional levels related to disease progression in prostate cancer were altered, which could have differed from the stromal changes noted in other cancer types ( 99 ). Notably, fibroblasts can differentiate into CAFs, which have been shown to play critical roles in tumor initiation and progression ( 99 ). The complexity of interactions between fibroblasts and PCa cells involves multiple signaling pathways, complicating our understanding of their interconnected relationships within the TME. To elucidate the potential interaction mechanisms between C3 NEFH+ malignant cells and fibroblasts, we employed a systematic analysis of intercellular communications using the CellChat tool. Our results revealed a robust communication network between C3 NEFH+ malignant cells and fibroblasts. Importantly, C3 NEFH+ malignant cells were found to secrete the factor PTN, which interacts with fibroblasts via its receptor, NCL. This interaction is pivotal in facilitating the transformation of fibroblasts into CAFs, a process closely associated with tumor progression. These findings underscore the significant role of C3 NEFH+ malignant cells in modulating the PCa microenvironment. The biological implications of our findings are profound. C3 NEFH+ malignant cells appear to actively reconfigure the stromal landscape, thereby creating an environment conducive to tumor growth and metastasis. This insight suggests that targeting C3 NEFH+ malignant cells may offer a promising therapeutic strategy. Disruption of the signaling pathways that mediate the interaction between these malignant cells and fibroblasts could lead to the development of novel treatments aimed at counteracting the supportive role of the stroma in tumor progression. From a therapeutic standpoint, targeting the PTN-NCL signaling axis may provide an innovative approach to inhibit CAF formation and thereby attenuate tumor aggressiveness. Such targeted therapies could be synergistically integrated into existing treatment protocols, potentially enhancing patient outcomes by addressing the TME’s influence on cancer progression. A key difficulty in drug development was ensuring the specificity and selectivity of the drug in order to reduce off-target effects. For instance, the knockdown of PTN in young mice resulted in defects in adult neurogenesis and cognitive dysfunction. This highlighted the need for careful consideration of the potential neurotoxic effects when developing drugs targeting the PTN-NCL signaling axis ( 100 ). Drug resistance was another significant challenge. In triple-negative breast cancer (TNBC) tissues that had relapsed after chemotherapy, the expression of PTN and its receptor PTPRZ1 was found to be elevated, which was closely linked to a poor prognosis ( 101 ). This suggested that tumors might acquire resistance by upregulating the PTN-NCL signaling axis during treatment, a factor that needed to be considered in drug development. Overall, these findings highlight the critical need for further research into the dynamic interactions within the TME and their implications for developing effective therapeutic interventions. Section title: Discussion Educational score: 4.205472946166992 Domain: biomedical Document type: Study Language: en In our in-depth analysis of TFs, we identified key TFs in the C3 NEFH+ malignant cell subtype, including NEUROD1, IRX4, LTF, POURA, and ELK4. Notably, IRX4 exhibited the highest subtype variance proportion in the M4 module, indicating its central regulatory role in C3 NEFH+ malignant cells. Moreover, IRX4 demonstrated high specificity scores in both the C3 NEFH+ malignant cell subtype and THG tissue, suggesting a strong regulatory relationship between this TF and its target genes. Consequently, IRX4 may serve as a potential biomarker or therapeutic target. Survival analysis results indicated that the expression level of IRX4 is likely associated with poorer prognosis (P<0.05). Previous research has confirmed that the knockdown of IRX4 can suppress stem-like characteristics and resistance to gefitinib in non-small cell lung cancer cells ( 102 ), although the specific mechanisms of IRX4 in PCa remain unclear. Thus, IRX4 may become a promising focal point in PCa research. Section title: Discussion Educational score: 4.073797225952148 Domain: biomedical Document type: Study Language: en Early and accurate identification of high-grade PCa is crucial for advancing both basic research and clinical practice. However, existing methods are limited due to the lack of sensitive and specific biomarkers. Previous studies have evaluated at least ten prognostic models based on various gene signatures and machine learning algorithms; however, these models typically perform poorly in survival prediction (AUC < 0.6) ( 103 ). To address this gap, we constructed a new predictive model based on the top 100 marker genes of the C3 subtype, achieving good predictive accuracy. The final five genes associated with poor prognosis were ZNF782, ZNF695, YY1, NR1I3 , and FOXA3 . These findings provide new directions for cancer prediction and diagnosis. Section title: Discussion Educational score: 4.570319652557373 Domain: biomedical Document type: Study Language: en Immune checkpoints play a critical role in regulating immune responses. Tumor cells often evade immune surveillance by upregulating immune checkpoints to inhibit local immune responses ( 104 ). Considering the widespread presence of immune cells in the TME of PCa, we analyzed differences in immune infiltration across different risk assessment categories. Compared to the low NmRS group, the high NmRS group exhibited significantly increased infiltration of naive B cells and resting CD4 memory T cells, likely reflecting an immune response associated with immune evasion ( 105 ). Interestingly, we found a negative correlation between the predictive model score and M1 and M2 macrophages, suggesting that tumors may promote the polarization of macrophages toward the M2 phenotype, thereby inhibiting M1 activity and leading to a dual reduction in the number of M1 and M2 macrophages. This change may be related to tumor progression and poor prognosis. Despite enhanced immune suppression and poorer prognosis in high-risk patient groups, they may exhibit greater sensitivity to chemotherapeutic agents such as Dasatinib and Bicalutamide, providing new research directions for subsequent clinical interventions. Tumor vaccines were found to have the potential to supplement conventional cancer therapies and targeted treatments. Their mechanism involved reprogramming the immune system to target and destroy cancer cells. These vaccines could have been one of the most promising strategies to overcome the inherent resistance in current cancer therapies ( 106 ). By examining the interactions between immune cells in the tumor microenvironment, new therapeutic approaches were developed, such as enhancing anti-tumor immune responses through modulation of the Th1/Th2 balance or influencing the function of Treg cells. The spatial architecture of the tumor microenvironment, particularly the tumor-stroma boundary, was emphasized for its impact on immune checkpoint blockade (ICB) efficacy. Targeting specific cell populations in defined spatial regions, like CXCL14+ CAFs, might have sensitized ICB responses ( 107 ). Although more research and clinical trials were needed to optimize immune microenvironment modulation for better therapeutic outcomes, the insights provided new avenues for developing treatments for PCa. Real-world studies (RWS) served as an important tool for assessing the efficacy and safety of drugs, especially in infectious diseases with multiple infection sites and complex pathogens. In the case of prostate cancer, this meant the need to evaluate the effectiveness of treatment regimens in a broader patient population ( 108 ). RWS showed that infections caused by multidrug-resistant Pseudomonas aeruginosa were difficult to treat, as many antibiotics were ineffective against this critical pathogen. This suggested that in prostate cancer, multidrug resistance (MDR) might also be a significant issue, highlighting the need for new treatment strategies to overcome it ( 109 ). Section title: Discussion Educational score: 4.43911075592041 Domain: biomedical Document type: Study Language: en Single-cell sequencing technology has transformed biological research by enabling the detailed analysis of individual cells. This advancement has provided critical insights into cellular heterogeneity and the complex molecular mechanisms underlying diseases like PCa. In the realm of personalized treatment, single-cell analysis opens new avenues for understanding the tumor microenvironment and identifying specific cell types or subpopulations that contribute to disease progression or therapeutic resistance. By allowing a more nuanced view of tumor heterogeneity, single-cell analysis enhances our understanding of PCa biology. The identification of the C3 NEFH+ malignant cell subtype, in particular, presents a valuable opportunity for future clinical research. Investigating this subtype could lead to the development of targeted approaches for early screening and the optimization of treatment strategies. This includes the identification of potential biomarkers to enhance detection capabilities and the recognition of specific therapeutic targets to improve treatment effectiveness. Furthermore, comprehending the unique characteristics of the C3 NEFH+ subtype will facilitate patient stratification and enable personalized treatment decisions, ultimately leading to better patient outcomes. However, this study has several important limitations. Firstly, the sample size is relatively small, focusing primarily on single-cell data from a subtype of PCa patients, which may restrict the generalizability and applicability of the results. Secondly, the analytical methods used in this study mainly relied on single-cell sequencing and transcriptomic analysis, without considering other factors that may influence the outcomes. Therefore, future research should conduct multicenter studies with larger sample sizes to validate the potential roles of IRX4 and the constructed prognostic model in PCa. Additionally, incorporating proteomics and metabolomics approaches will provide deeper insights into the functional characteristics of specific subgroups and key molecules, thereby offering a more comprehensive basis for the early diagnosis and individualized treatment strategies for PCa. Through multi-omics analysis, we can better understand the biological mechanisms of tumors and identify potential therapeutic targets. In summary, our research focused on the diversity of epithelial cells in high-grade PCa at the individual cell level, further revealing the significance of IRX4 in this cancer type. Moreover, we identified several prognostically relevant genes, discovering a significant correlation between a higher NmRS and poorer prognosis. These findings not only enhance our understanding of the developmental mechanisms of PCa but also provide new opportunities for predicting and diagnosing this disease, with important clinical implications. Future studies should continue to explore these discoveries to advance research and treatment progress in PCa. Section title: Conclusion Educational score: 4.156330108642578 Domain: biomedical Document type: Study Language: en This study utilized technology to delve into the complexities of the PCa TME, revealing the interactions between various cellular components and their influence on tumor progression. We identified the C3 NEFH+ malignant cell subtype, which was associated with high-grade PCa and exhibits increased malignancy. The communication between malignant epithelial cells and fibroblasts through the PTN signaling pathway may be linked to the transformation of CAFs. TFs analysis identified key regulators, such as IRX4, which plays a central role in C3 NEFH+ cells, with its expression level significantly correlating with patient prognosis. These findings provide new biomarkers and therapeutic targets for the early diagnosis and treatment of PCa. Furthermore, our survival prediction model based on C3 subtype marker genes demonstrated promising results, offering a new tool for clinical practice. Section title: Conclusion Educational score: 3.8561623096466064 Domain: biomedical Document type: Review Language: en In summary, this research enhances our understanding of the PCa microenvironment and lays the groundwork for future therapeutic strategies and biomarker development. We anticipate that subsequent studies will validate these findings and explore their potential clinical applications, particularly focusing on cellular heterogeneity within the tumor microenvironment, elucidation of resistance mechanisms, and the development of early diagnostic biomarkers, thereby advancing our understanding of tumor biology. | Review | biomedical | en | 0.999997 |
PMC11695426 | Section title: Introduction Educational score: 3.931415319442749 Domain: biomedical Document type: Study Language: en Adolescent aggression is a pressing social issue that is both prevalent and profoundly impacts the individuals involved ( 1 ). A survey of approximately 80,000 adolescents revealed that 18% reported engaging in aggressive behavior ( 2 ). For adolescents who perpetrate aggression, these experiences can lead to a cascade of negative consequences, such as substance abuse and depressive tendencies ( 3 ). Fite et al. ( 4 ) found that adolescents with a history of aggression often exhibit greater levels of anxiety and more antisocial behavior in adulthood. Meanwhile, adolescents who are victimized by aggression may experience anxiety, depression, and other psychological problems; develop fears of interpersonal relationships and trust issues; and may even suffer from long-term psychological trauma. Such psychological issues not only impact the academic performance and social functioning of these individuals but may also persist into adulthood, leading to mental health problems and social difficulties ( 5 ). Adolescence, particularly secondary school period, is a critical period for the development and expression of aggressive behavior ( 6 ). Chen et al. ( 7 ) analyzed the changes in aggression patterns that occur in the transition from eighth to ninth grade and found that the proportion of low-aggression adolescents who become high-aggression adolescents was as high as 28.8%, highlighting the importance of early identification and intervention. Section title: Introduction Educational score: 3.8216607570648193 Domain: biomedical Document type: Review Language: en Based on the functions of aggression and the emotional involvement of the aggressor, aggressive behavior can be categorized as either reactive or proactive aggression ( 8 ). Reactive aggression is impulsive, triggered by negative emotions such as anger or frustration, and serves as emotional release. It often stems from cognitive biases, where individuals with limited cognitive skills may be prone to hostile attribution errors, perceiving threats and reacting aggressively ( 9 ). This type of aggression is prevalent in emotionally strained environments, such as in families with poor communication or high parental criticism, where adolescents express distress through reactive aggression ( 10 , 11 ). In contrast, proactive aggression is planned, unemotional, and goal-driven, typically occurring in contexts of power struggles or competition, such as schools where individuals seek social status or material rewards ( 12 , 13 ). Overall, reactive aggression is common in emotionally charged situations, while proactive aggression is more likely to arise in strategic, competitive environments. Section title: Introduction Educational score: 2.2050256729125977 Domain: biomedical Document type: Study Language: en Given the differences in behavioral responses and cognitive processes between reactive and proactive aggression, this study, focusing on Chinese adolescents, sought to examine the underlying mechanisms of both types of aggression. Section title: Executive dysfunction and aggressive behavior Educational score: 4.260387897491455 Domain: biomedical Document type: Study Language: en Adolescence is a peak period for the onset of aggressive behavior. During this developmental stage, individuals’ executive functions, which represent a set of cognitive skills that involve top-down control processes elicited in the planning, organizing, and monitoring of complex, goal-directed behavior ( 14 ), may lag behind the demands of their environment, making it difficult for them to accurately perceive events and regulate intense aggressive emotions ( 15 ). A longitudinal study by Maloney et al. ( 16 ) found that adolescents with executive dysfunction exhibited more aggressive behavior in adulthood. Individuals with lower levels of executive function (cognitive ability) are more likely to interpret others’ intentions as hostile in ambiguous situations ( 17 ), and they tend to develop hostile attribution biases during the stages of cue encoding and interpretation, which can lead to aggressive behavior in the final stage of behavioral execution ( 9 ). Additionally, Wood and Worthington ( 18 ) suggested that damage to brain regions associated with executive function, such as the dorsolateral prefrontal cortex, orbitofrontal cortex, and ventromedial prefrontal cortex, can impair individuals’ ability to regulate emotions, make decisions, and control impulses. They also noted that executive function primarily develops during adolescence, which means that any damage during this critical period can have significant long-term effects on these abilities ( 18 ). Therefore, the level of executive function is not only related to cognitive processes but also closely linked to emotion regulation and behavioral control. These factors together may lead to individuals’ misinterpretation of social cues and intensified emotional responses, ultimately manifesting as aggressive behavior. Section title: Executive dysfunction and aggressive behavior Educational score: 4.15657901763916 Domain: biomedical Document type: Study Language: en Previous studies have often examined aggressive behavior as a whole, with fewer studies differentiating between different types of aggressive behavior and looking at their relationship with executive dysfunction. In fact, there is evidence suggesting that the impact of executive dysfunction on different subtypes of aggression may vary ( 19 ). Proactive aggression is a premeditated, goal-directed behavior, typically undertaken to achieve a specific purpose. Executive function impairments may affect individuals’ ability to plan, organize, and make decisions. Damage to the dorsolateral prefrontal cortex may impair individuals’ “cold” executive functions, including logical reasoning and problem-solving ( 20 ), which may affect a person’s ability to plan their aggressive actions to solve problems. Reactive aggression, on the other hand, is an immediate response to social threats, provocations, or frustrations, and it is often accompanied by strong emotional reactions. Impairments in “hot” executive functions may affect individuals’ ability to regulate emotions; for example, damage to the ventromedial prefrontal cortex and orbitofrontal cortex may impair individuals’ processing of emotionally salient information, including empathy and social judgment, which may lead to hypersensitivity to provocation and an inability to appropriately regulate emotional responses ( 21 ). Section title: Executive dysfunction and aggressive behavior Educational score: 4.009912014007568 Domain: biomedical Document type: Study Language: en Thomson and Centifanti ( 22 ) measured individuals’ executive functions and found that performance on executive function tasks (such as the Stroop task) significantly predicted both proactive and reactive aggression, with a stronger association between reactive aggression and executive function. These conclusions were verified in a 3-year longitudinal study by Rohlf et al. ( 23 ), who found that early childhood executive dysfunction positively predicted later childhood proactive and reactive aggression and that the correlation between executive dysfunction and reactive aggression was stronger than that involving proactive aggression. Based on existing theories and empirical research, the current study proposes the following two hypotheses: (1a) executive dysfunction significantly positively predicts proactive aggression and (1b) executive dysfunction significantly positively predicts reactive aggression, with a stronger correlation between adolescents’ executive dysfunction and reactive aggression. Section title: The mediating role of impulsivity Educational score: 4.064327239990234 Domain: biomedical Document type: Review Language: en Impulsivity is a broad construct that has multiple operationalizations. Definitions include risk-taking, sensation-seeking, behavioral disinhibition, preference for small immediate rewards over large distal rewards, deficits in planning, and urgency ( 24 ). This trait is particularly prevalent among adolescents, who often respond to internal and external stimuli in a hasty and unplanned manner ( 25 ). Recent research has shed light on the intricate relationship between executive function, impulsivity, and aggressive behavior. Finkel and Hall ( 26 ) proposed the I 3 model of aggression, suggesting that aggressive behavior arises from the interaction of personality traits like impulsivity, self-regulatory capacities such as executive function, and external environmental factors. Impairments in executive function can lead to deficits in cognitive processes involved in evaluating the consequences of behavior, increasing impulsivity—the tendency to act without fully considering potential negative outcomes ( 27 ). Section title: The mediating role of impulsivity Educational score: 4.061100959777832 Domain: biomedical Document type: Study Language: en This relationship has also been empirically supported in behavioral experiments. Reynolds et al. ( 28 ) measured participants’ executive function using the Wisconsin Card Sorting Test and assessed their aggressive behavior and impulsivity using questionnaires. They found that participants’ executive function was negatively correlated with impulsivity and that participants’ impulsivity positively predicted their own aggressive behavior. Notably, impulsivity’s influence on aggression may vary depending on the subtype of aggression. Research indicates that impulsivity primarily predicts reactive aggression rather than proactive aggression ( 29 ), a conclusion echoed by Duan et al. ( 30 ), who found that trait impulsivity was significantly related to reactive aggression in a competitive reaction time task. Curtis et al. ( 31 ) further explored this relationship, identifying a significant conceptual and cognitive overlap between proactive and reactive aggression, suggesting that the distinction between the two types may not be as pronounced in the context of impulsivity. Section title: The mediating role of impulsivity Educational score: 4.084400177001953 Domain: biomedical Document type: Study Language: en Given the controversy surrounding the relationship between impulsivity and subtypes of aggression and the fact that previous cross-sectional studies could not reveal causal relationships, the present study examines the mediating role of impulsivity in the relationship between executive dysfunction and aggressive behavior using a longitudinal design, while differentiating between proactive and reactive aggression. Based on this, the present study proposes the following two additional hypotheses: (2a) impulsivity mediates the effect of executive dysfunction on proactive aggression, (2b) impulsivity also mediates the relationship between executive dysfunction and reactive aggression, and (2c) the mediating effect of impulsivity is more pronounced in the context of reactive aggression compared to proactive aggression. Section title: The moderating role of sex Educational score: 4.080889701843262 Domain: biomedical Document type: Study Language: en When discussing the relationship between impulsivity and aggressive behavior, sex is an important factor to consider. Previous research has shown that sex is closely related to aggressive behavior, with males generally exhibiting higher levels of aggression compared to females ( 32 ), especially in impulsive aggression ( 33 ). This difference can be partly explained by physiological factors, including neurotransmitters such as serotonin, which is crucial for emotional regulation and impulse control ( 34 ). Males have been found to have lower serotonin regulation abilities when faced with stress or provocation, leading to weaker impulse control and greater difficulty managing aggression ( 35 ). Additionally, testosterone, the primary male sex hormone, has been linked to increased aggression and dominance behavior, especially in competitive or resource-driven situations ( 36 ). Testosterone’s effects on brain regions involved in emotion and impulse regulation, such as the prefrontal cortex and amygdala, may further explain why males are more prone to impulsive aggression in response to provocation ( 36 , 37 ). Section title: The moderating role of sex Educational score: 3.840096950531006 Domain: biomedical Document type: Study Language: en While both executive dysfunction and impulsivity contribute to aggression, the moderating role of sex is more relevant to impulsivity than to executive function. This is because physiological factors, such as serotonin and testosterone levels, differ between males and females and directly affect impulsive behavior and aggression. In contrast, executive dysfunction is linked to cognitive control processes, which are less influenced by these biological differences ( 38 ). Therefore, when examining aggression, it is more important to consider how sex influences impulsivity, as males’ biological makeup makes them more prone to impulsive aggression. Section title: The moderating role of sex Educational score: 4.011303901672363 Domain: biomedical Document type: Study Language: en Building on this, Dinić and Wertag ( 39 ) explored the relationship between different subtypes of aggression and sex, finding that males scored significantly higher than females in both proactive and reactive aggression. Specifically, males were more likely to exhibit reactive aggression under the same level of anger ( 40 ). The connection between impulsivity and reactive aggression is particularly strong in males, as their relatively weaker impulse control makes it more difficult to suppress aggressive responses when provoked or stressed ( 41 ). This is especially evident among male juvenile delinquents ( 42 ). In contrast, females’ aggressive behavior tends to be shaped more by social and environmental factors, such as early trauma or neglect, which are more strongly associated with proactive aggression in females ( 43 , 44 ). Additionally, impulsivity in females has been linked to mental health problems, including suicidal behavior, underscoring the importance of early intervention in this population ( 45 ). Section title: The moderating role of sex Educational score: 3.969839572906494 Domain: biomedical Document type: Study Language: en In summary, physiological differences in impulse control, as well as psychological factors like early experiences and communication skills, work together to influence the expression of aggressive behavior. Males may be more inclined to react immediately to external stimuli, while females may be more likely to transform internal emotions into proactive aggression. These findings collectively emphasize the importance of considering sex differences and related psychosocial factors in the prevention and intervention of adolescent aggressive behavior. Based on these findings, this study proposes hypothesis 3a: sex moderates the effect of impulsivity on proactive aggression. This study also proposes hypothesis 3b: sex moderates the effect of impulsivity on reactive aggression, as detailed in the proposed model in Figure 1 . Section title: Participants Educational score: 2.5909817218780518 Domain: biomedical Document type: Study Language: en This study employed a cluster sampling method, targeting middle school students from Sichuan Province, China. A total of 630 students participated, and the final sample comprised 617 valid questionnaires, with a response rate of 97.93%. The average age of the participants was 15.26 years (SD = 1.37), with an age range of 13 to 17 years. Among the participants, 253 were female (41.0%) and 364 were male (59.0%). Section title: Reactive–proactive aggression questionnaire Educational score: 3.921030044555664 Domain: biomedical Document type: Study Language: en The Reactive–Proactive Aggression Questionnaire developed by Raine et al. ( 46 ) was used to measure participants’ reactive and proactive aggression. This questionnaire consists of 25 items, of which 13 items measure reactive aggression (e.g., “Yelled at others when they have annoyed you”) and 12 items measure proactive aggression (e.g., “Hurt others to win a game”). The questionnaire used a 3-point Likert scale, with 0 points representing “never,” 1 point representing “sometimes,” and 2 points representing “often.” The total score of the scale was calculated, with higher scores indicating greater levels of reactive or proactive aggression. This study used the Chinese version revised by You et al. ( 47 ). In this study, the internal consistency was assessed using Cronbach’ s α. At T1, Cronbach’s α was 0.756 for reactive aggression and 0.651 for proactive aggression. At T2, these values increased to 0.807 and 0.734, respectively, indicating satisfactory to good internal consistency for both reactive and proactive aggression scales at both time points. Section title: Teenage executive functioning inventory Educational score: 4.032887935638428 Domain: biomedical Document type: Study Language: en The Teenage Executive Functioning Inventory, developed by Thorell et al., was used to assess adolescents' deficits in working memory and inhibitory control ( 48 ). The questionnaire consists of 20 items, of which 11 items measure working memory (e.g., “Has difficulties with tasks involving several steps that need to be completed in a certain order”) and nine items measure inhibition (e.g., “ Puts things off until the last minute”). The questionnaire uses a 5-point Likert scale, with scores ranging from 1 point (“absolutely not”) to 5 points (“absolutely yes”). The total score of the scale was calculated, with higher scores indicating more severe executive dysfunction. The scale has good reliability and validity ( 48 ). This study used the Chinese version revised by Hu et al. ( 49 ). In this study, the internal consistency coefficients of the scale at T1 and T2 were 0.891 and 0.898, respectively. Section title: Barratt impulsiveness scale Educational score: 3.9812023639678955 Domain: biomedical Document type: Study Language: en The Barratt Impulsiveness Scale ( 50 ) was used to assess trait impulsivity. This questionnaire consists of 30 items and uses a 5-point Likert scale for responses, encompassing three dimensions: motor impulsivity, cognitive impulsivity, and non-planning impulsivity. Each dimension contains 10 items, such as “I do things without thinking” for motor impulsivity, “I make-up my mind quickly” for cognitive impulsivity, and “I plan tasks carefully” for non-planning impulsivity. The total score of the questionnaire was calculated for each participant, with higher scores indicating greater levels of impulsivity. This study used the Chinese version revised by Li et al. ( 51 ). In this study, the internal consistency coefficients of the scale at T1 and T2 were 0.878 and 0.888, respectively. Section title: Research procedures and statistical analysis Educational score: 2.0293428897857666 Domain: biomedical Document type: Study Language: en Data collection was carried out at two time points: April 2023 (T1) and April 2024 (T2). The questionnaires were administered by trained psychology teachers and group activity organizers and were collected on-site by the assessors. Prior to the study, informed consent was obtained from all participants and their guardians. Each participant and guardian received a comprehensive description of the study procedures, including the questionnaire and any associated precautions. This information was provided by the principal investigator to ensure that all parties were fully informed before participation. This study has been approved by the Ethics Committee of Sichuan Vocational and Technical College , in accordance with the Declaration of Helsinki. Section title: Research procedures and statistical analysis Educational score: 4.0596923828125 Domain: biomedical Document type: Study Language: en A longitudinal research design was adopted to delve into the association between executive dysfunction and aggressive behavior in adolescents, as well as to examine the mediating role of impulsivity and the moderating role of sex in this relationship. We chose a longitudinal study design for two reasons: first, adolescence is a critical period for the development of aggressive behavior, and there is substantial evidence of significant shifts in adolescent aggression during this period ( 6 , 52 ), highlighting the need for longitudinal studies to capture behavioral changes over time; second, longitudinal research can reveal temporal sequences among variables, providing more rigorous evidence for causal inferences. Section title: Research procedures and statistical analysis Educational score: 3.91494083404541 Domain: biomedical Document type: Study Language: en Descriptive statistics and correlation analyses were conducted using SPSS version 26.0 (IBM Corporation, Armonk, NY, USA). To examine the mediating role of impulsivity, we applied Model 4 of the PROCESS macro ( 53 ). This model assesses whether impulsivity mediates the relationship between executive dysfunction and aggression. Additionally, to explore the potential moderating effect of sex on the mediating relationship, we utilized Model 14 of the PROCESS macro. This model allowed us to assess whether the mediating effect of impulsivity on the relationship between executive dysfunction and aggression varies by sex. All mediation assumptions were met, with significant correlations supporting the validity of the analyses. Section title: Common method bias Educational score: 3.6827738285064697 Domain: biomedical Document type: Study Language: en To address potential common method bias, Harman’s single-factor test was conducted on the self-reported data. The results showed that, at T1, there were 18 factors with eigenvalues greater than 1, and the first factor explained 17.52% of the total variance, which was below the critical value of 40%. At T2, there were 16 factors with eigenvalues greater than 1, and the first factor explained 19.06% of the total variance, thus also being below the critical value of 40%. Therefore, the data in this study did not have a serious common method bias problem ( 54 ). Section title: Descriptive and correlation analysis Educational score: 2.6568949222564697 Domain: biomedical Document type: Study Language: en Table 1 presents the means of the variables involved in this study. The results showed that there were no significant differences between the two measurements for all variables except proactive aggression. Overall, there were no significant differences between the two measurements. Subsequently, correlation analyses were conducted on executive dysfunction, impulsivity, and reactive and proactive aggression at T1 and T2, and the results showed that there were significant positive correlations between all pairs of variables, as seen in Table 1 . Section title: Tests of mediating effect of executive dysfunction Educational score: 4.106372833251953 Domain: biomedical Document type: Study Language: en First, Model 4 of Hayes ( 53 ) PROCESS macro was used to examine the mediating effect of impulsivity on reactive aggression . Regression analysis was conducted with T1 executive dysfunction as the independent variable and T2 reactive aggression as the dependent variable. The results showed that T1 executive dysfunction significantly and positively predicted T2 reactive aggression ( β = 0.34, t = 9.02, p < 0.001). After adding the mediating variable T2 impulsivity, the direct effect of T1 executive dysfunction on T2 reactive aggression remained significant ( β = 0.21, t = 5.38, p < 0.001), while the predictive effect of T1 executive dysfunction on T2 impulsivity was significant ( β = 0.41, t = 11.32, p < 0.001) and the predictive effect of T2 impulsivity on T2 reactive aggression was significant ( β = 0.31, t = 7.71, p < 0.001), respectively. The 95% confidence interval (CI) of the mediating effect of T2 impulsivity between T1 executive dysfunction and T2 reactive aggression was 0.08–0.17 (Sobel test = 6.38 SE = 0.02, p < 0.01), indicating a significant partial mediating effect. The mediating effect accounted for 38% of the total effect (total effect = 0.34, direct effect = 0.21). Section title: Tests of mediating effect of executive dysfunction Educational score: 4.07904052734375 Domain: biomedical Document type: Study Language: en Similarly, the mediating effect of impulsivity on proactive aggression was examined . Regression analysis was conducted with T1 executive dysfunction as the independent variable and T2 proactive aggression as the dependent variable. The results showed that T1 executive dysfunction significantly and positively predicted T2 proactive aggression ( β = 0.22, t = 5.58, p < 0.001). After adding the mediating variable T2 impulsivity, the direct effect of T1 executive dysfunction on T2 proactive aggression remained significant ( β = 0.13, t = 3.11, p < 0.01); also, the predictive effect of T1 executive dysfunction on T2 impulsivity was significant ( β = 0.41, t = 11.27, p < 0.001) and the predictive effect of T2 impulsivity on T2 proactive aggression was significant ( β = 0.21, t = 4.99, p < 0.001). The 95% CI of the mediating effect of T2 impulsivity between T1 executive dysfunction and T2 proactive aggression was 0.04–0.12 (Sobel test = 4.90 SE = 0.01, p < 0.01), indicating a significant partial mediating effect. The mediating effect accounted for 36% of the total effect (total effect = 0.22, direct effect = 0.13). Section title: Test of moderated mediation model effect Educational score: 3.8536434173583984 Domain: biomedical Document type: Study Language: en Model 14 of Hayes ( 53 ) PROCESS macro was used to analyze the moderated mediation model with T2 reactive aggression as the dependent variable. The results showed that sex moderated the latter half of the mediation path ( β = 0.16, SE = 0.07, p = 0.025, 95% CI [0.02–0.31]) (total effect = 0.28, direct effect = 0.21). The mediating effect value for the male group was 0.15, while that for the female group was 0.09. Section title: Test of moderated mediation model effect Educational score: 3.812403678894043 Domain: biomedical Document type: Study Language: en Further simple slope analysis showed that T2 impulsivity had a significant positive predictive effect on T2 reactive aggression in the male group ( β = 0.38, p < 0.001), while T2 impulsivity in the female group had a significant but smaller positive predictive effect on T2 reactive aggression ( β = 0.21, p < 0.001). Section title: Test of moderated mediation model effect Educational score: 3.724961280822754 Domain: biomedical Document type: Study Language: en Similarly, with T2 proactive aggression as the dependent variable, Model 14 of Hayes ( 53 ) PROCESS macro was used to analyze the moderated mediation model. The results showed that sex moderated the latter half of the mediation path ( β = 0.20, SE = 0.07, p = 0.007, 95% CI [0.05–0.36]) (total effect = 0.21, direct effect = 0.13). The mediating effect value for the male group was 0.12, while that for the female group was not significant at 0.03 (p > 0.05). Section title: Test of moderated mediation model effect Educational score: 3.5906598567962646 Domain: biomedical Document type: Study Language: en Further simple slope analysis showed that T2 impulsivity had a significant positive predictive effect on T2 proactive aggression in the male group ( β = 0.30, p < 0.001), while T2 impulsivity in the female group did not have a significant positive predictive effect on T2 proactive aggression ( β = 0.09, p > 0.05). Section title: Discussion Educational score: 3.8381009101867676 Domain: biomedical Document type: Study Language: en This study investigated the longitudinal relationship between executive dysfunction and adolescent proactive and reactive aggression and explored the potential mechanisms and sex differences in the relationship between executive dysfunction and aggressive behavior based on the mediating variable of impulsivity. Section title: The impact of executive dysfunction on reactive and proactive aggression Educational score: 4.107538223266602 Domain: biomedical Document type: Study Language: en The results of this study are consistent with those of previous research ( 23 ), indicating that prior executive dysfunction significantly and positively predicts both reactive and proactive aggression among Chinese adolescents, supporting hypotheses 1a and 1b. Executive dysfunction has long been associated with various forms of aggression, including proactive and reactive aggression ( 55 ). Specifically, reactive aggression, often driven by frustration and emotional responses, has been shown to have a stronger association with executive dysfunction than proactive aggression. Hu et al. ( 56 ) highlighted that adolescents with executive dysfunction are more prone to reactive aggression, resorting to retaliatory behavior after experiencing negative emotions. This is consistent with findings from Tonnaer et al. ( 55 ), which suggest that impairments in response inhibition—a key component of executive function—are stronger predictors of reactive aggressive behavior than other executive capacities.Proactive aggression, in contrast, involves planned and goal-directed behavior that serves a strategic purpose ( 12 ). While it requires some degree of planning and self-regulation, previous research has found that deficits in these areas of executive function, such as planning and organizational abilities, may impair an individual’s capacity for proactive aggression ( 22 ). In this study, however, executive dysfunction was found to predict both reactive and proactive aggression, suggesting a more generalized impact on aggressive behavior, beyond the differences between reactive and proactive subtypes. Section title: The impact of executive dysfunction on reactive and proactive aggression Educational score: 4.132951736450195 Domain: biomedical Document type: Study Language: en Interestingly, contrasting with our results, Hecht and Latzman ( 57 ) found that proactive aggression is associated with higher levels of working memory, which is a core component of executive function. The participants in this study were middle school students, while the participants in the study by Hecht and Latzman ( 57 ) were college students. Such a difference in age may be one reason for the inconsistent results. Similar to the results of this study, Jakubovic and Drabick ( 58 ) found in their investigation that lower working memory was associated with greater proactive aggression in adolescents. Working memory undergoes reorganization in brain-processing functions at the onset of puberty, typically around adolescence ( 59 ). With age, the development of working memory shifts from the activation of visuo-spatial or motor networks to the activation of executive networks ( 60 ). This change may mean that there are differences in the mechanisms underlying proactive aggression between adolescents and adults ( 61 ). In addition, response inhibition is a major predictor of aggressive behavior ( 62 ). The inhibition of executive function may not only affect the impulsive and aggressive behavior exhibited in response to provocation or conflict in reactive aggression ( 21 , 57 ) but also influence individuals’ over-positive evaluation of the consequences of aggressive behavior in proactive aggression. Therefore, executive dysfunction can also positively predict proactive aggression in adolescents. Section title: Differences in the predictive levels of impulsivity for reactive and proactive aggression Educational score: 3.897153615951538 Domain: biomedical Document type: Study Language: en The link between impulsivity and aggressive behavior has been well-established in numerous studies ( 63 – 65 ), and other research has found that impulsivity may have different relationships with different subtypes of aggression ( 66 ). This study further verified this point through a comparison of correlation coefficients. The results showed that impulsivity had a stronger predictive effect on reactive aggression than on proactive aggression. This is consistent with findings of previous research ( 29 , 30 ) indicating that impulsivity has a greater influence on individuals’ impulsive behavior when they are faced with provocation, while its predictive effect on planned proactive aggression is relatively weaker. Section title: Differences in the predictive levels of impulsivity for reactive and proactive aggression Educational score: 3.9156267642974854 Domain: biomedical Document type: Study Language: en Reactive aggression and proactive aggression, as two subtypes of aggression, share a significant amount of conceptual and cognitive overlap, and Curtis et al. ( 31 ) argued that there is no significant difference between these two types of aggression. However, the results of this study and other previous studies show that the effects of impulsivity on reactive and proactive aggression are different. In the case of provocation, impulsivity is a key predictor of high levels of retaliation (reactive aggression), and its role is more important than those of other forms of self-control ( 67 ). Greater levels of impulsivity are also associated with increased reactive aggression, both at the initial level and in longitudinal processes over time, with a close link present between impulsivity and reactive aggression ( 68 ). Proactive aggression, as a planned and unemotional form of aggression, is more influenced by cold-hearted traits and self-aggression associations than impulsivity ( 29 , 69 ). Section title: The moderating role of sex in the relationship between impulsivity and aggressive behavior Educational score: 3.9724695682525635 Domain: biomedical Document type: Study Language: en The findings of this study are consistent with those of Dinić and Wertag ( 39 ), indicating that impulsivity has a stronger predictive effect on proactive aggression in male adolescents than female ones. This finding confirms the significant moderating role of sex in the relationship between impulsivity and proactive aggressive behavior, supporting hypothesis 3a. The I 3 model of aggression suggests that aggressive behavior arises from the interaction of personality traits like impulsivity, self-regulatory capacities like executive function, and external environmental factors ( 26 ). This theory provides a framework for understanding differences in the expression of aggressive behavior between sexs. In social environments, individuals of different sexs not only have physiological differences but also experience different social lives. Section title: The moderating role of sex in the relationship between impulsivity and aggressive behavior Educational score: 4.1319684982299805 Domain: biomedical Document type: Study Language: en Proactive aggression, as a planned and unemotional behavior, is influenced by individuals’ sensitivity to rewards and punishments ( 12 ), and this sensitivity is significantly different between sexs. Bresin ( 66 ) pointed out that males are more sensitive to seeking stimulation and rewards, while females are more sensitive to punishment. In addition to the physiological influences of serotonin and testosterone, this difference can also be explained in the I 3 theory as an interaction between sex and the external environment. Due to the encouragement of direct and competitive behavior in the socialization process, males show greater sensitivity to impulsivity ( 70 ), and their degree of impulsivity is generally higher than that of females ( 71 ). These physiological and socialization tendencies may make males more likely to resort to aggressive behavior as a response when faced with impulsive challenges. In contrast, females’ impulsivity may be inhibited by socialization factors in the context of proactive aggression ( 44 ). Societal expectations of female behavior, such as maintaining harmonious interpersonal relationships, may reduce their impulsive expression of aggressive behavior. This socialization expectation constitutes an important factor in the external environment, demonstrating the important influence of social factors of sex on aggressive behavior. Section title: The moderating role of sex in the relationship between impulsivity and aggressive behavior Educational score: 3.873347282409668 Domain: biomedical Document type: Study Language: en Unlike proactive aggression, simple slope tests revealed that impulsivity is a strong predictor of reactive aggression for both boys and girls, with a more significant effect observed in boys. This finding is supported by Vaughan et al. ( 68 ), who demonstrated that the relationship between impulsivity and reactive aggression symptoms was notable in both sexes. However, the study indicated that the predictive power of impulsivity for reactive aggression is greater in males compared to females, which reflects the distinct characteristics of aggression subtypes across genders ( 72 ). Additionally, it highlights the complex interplay between cognitive processes and behavior ( 19 ). Section title: The moderating role of sex in the relationship between impulsivity and aggressive behavior Educational score: 2.932762861251831 Domain: other Document type: Study Language: en This pattern aligns with findings from Cano-Lozano et al. ( 73 ), which indicated that boys are more likely to exhibit reactive violence toward fathers in response to parental victimization, while girls demonstrated more reactive violence toward both parents in various victimization contexts. Furthermore, Navas-Martínez and Cano-Lozano ( 74 ) emphasized that girls in the generalist profile tend to engage in psychological and control/domain violence toward mothers, suggesting that their aggression is often reactive to familial dynamics. In contrast, boys, who are found to exercise more physical violence, may be socialized to display more overtly aggressive behaviors. This divergence may be rooted in the different socialization processes that influence how boys and girls learn to respond to conflict, thus reinforcing the notion that impulsivity manifests differently across genders. Section title: The moderating role of sex in the relationship between impulsivity and aggressive behavior Educational score: 3.726027011871338 Domain: biomedical Document type: Study Language: en Taken together, the I 3 model offers a multidimensional framework that allows for a deeper understanding of how sex, as part of the external environment, influences individuals’ impulsivity and aggressive behavior ( 26 ). Sex differences are not only reflected at the physiological level but are also deeply rooted in the socio-cultural context and socialization process. By utilizing this model, we can consider personality traits, self-regulatory capacities, external environmental factors, and their interactions to more comprehensively analyze and predict the occurrence of aggressive behavior. This approach can also inform the development of effective intervention strategies to reduce aggressive behavior. Section title: Practical implications Educational score: 2.3003036975860596 Domain: biomedical Document type: Study Language: en In terms of practical implications, the findings of this study suggest that we should pay close attention to the impact of executive dysfunction, impulsive personality traits, and sex on adolescent aggressive behavior and give them due consideration when developing prevention strategies. Section title: Practical implications Educational score: 3.9953691959381104 Domain: biomedical Document type: Review Language: en Research has shown that executive dysfunction, especially that related to behavioral disinhibition, is significantly prevalent among adolescents with antisocial behavior ( 75 ). These adolescents are more likely to engage in impulsive and risky behaviors ( 28 ), including substance abuse, gambling, and aggression ( 76 ). Fortunately, executive function can be improved through training and intervention ( 77 ), and adolescence is a critical period for prevention and intervention of executive dysfunction ( 78 ). Schools and communities can implement effective intervention measures during this period, with a particular focus on the role of executive dysfunction and impulsivity in adolescent aggressive behavior, such as fostering students’ socio-emotional learning skills, teaching them how to recognize and regulate emotions, build positive relationships, manage conflict, and make responsible decisions ( 79 ). Clinicians can also design targeted interventions, such as cognitive–behavioral therapy, based on a deep understanding of the mediating role of impulsivity in the relationship between executive dysfunction and aggressive behavior. This will effectively help adolescents identify external cues, manage their emotions, learn more adaptive behaviors, and reduce impulsive aggression ( 7 ). Section title: Practical implications Educational score: 3.968414783477783 Domain: biomedical Document type: Study Language: en In addition, sex-differentiated intervention strategies cannot be ignored. The results of this study revealed a significant moderating effect of sex on the relationship between impulsivity and aggression. Impulsivity significantly mediates the relationship between executive dysfunction and both proactive and reactive aggression in male groups. However, impulsivity does not have a significant mediating effect on proactive aggression in females, which suggests that there may be social factors beyond impulsivity that influence proactive aggression in females. Modern intervention methods emphasize the importance of sex-sensitive interventions ( 39 ). When developing intervention strategies, we must recognize the impact of sex differences on adolescent aggressive behavior and design differentiated approaches accordingly. Section title: Practical implications Educational score: 3.908464193344116 Domain: biomedical Document type: Study Language: en For male adolescents, the strong predictive role of impulsivity in aggressive behavior suggests that interventions should focus on impulse control and emotion regulation. Impulse control training can teach them self-regulation strategies during emotional arousal ( 42 ). For example, anger control training can reduce impulsivity and aggression through relaxation techniques like deep breathing and problem-solving strategies. Lei et al. ( 80 ) found that self-control strategies effectively reduced aggressive behavior among suspended adolescents. Additionally, emotion regulation training can help male adolescents manage negative emotions, such as anger, and respond more appropriately in emotionally charged situations. In contrast, female adolescents’ proactive aggression may be more influenced by social role expectations, so interventions should emphasize social-emotional learning. This can help develop empathy and communication skills, enabling non-violent conflict resolution ( 44 ). Shechtman ( 81 ) showed that short-term multidimensional interventions, incorporating bibliotherapy, effectively reduced aggression and improved emotional regulation and social skills. These interventions can promote healthier coping strategies in social contexts for female adolescents. Section title: Limitations Educational score: 4.136893272399902 Domain: biomedical Document type: Study Language: en Although this study provides an understanding of the role of executive dysfunction, impulsivity, and sex in adolescent aggressive behavior, there are still some limitations. First, the study mainly focuses on individual factors, such as impulsivity and sex, while environmental variables—specifically, parental influence and peer relationships—remain unexamined. Research indicates that parental styles, such as authoritarian or permissive approaches, can significantly affect adolescents’ behavioral outcomes ( 82 ). Similarly, peer relationships can either mitigate or exacerbate aggressive behaviors, depending on the nature of those interactions ( 83 , 84 ). By failing to account for these critical environmental factors, the current study may miss key contextual influences that shape adolescent behavior.Furthermore, acknowledging these unmeasured variables is essential, as they may serve as potential confounders that could distort the study’s findings. For instance, high levels of impulsivity may be exacerbated by negative peer influences or inadequate parental support, which in turn could contribute to increased aggression. Future research should incorporate these environmental factors to provide a more nuanced understanding of the dynamics at play. Second, the reliance on self-reported data in this study introduces potential subjective bias, limiting the objectivity and accuracy of the results. Future research could enhance reliability by triangulating data from multiple sources, such as teacher ratings or peer nominations ( 85 ), which would provide a more comprehensive view of adolescent behavior. Finally, the one-year interval between measurements constrains the ability to fully explore the long-term predictive effects of executive dysfunction on aggressive behavior. Employing more complex statistical methods, such as latent growth modeling, could elucidate the long-term dynamics between these variables ( 68 ), ultimately offering stronger evidence for long-term interventions in adolescent mental health.By overcoming these limitations, future research will be able to provide deeper insights and offer more precise guidance for adolescent mental health and behavioral development. Section title: Conclusions Educational score: 3.7768332958221436 Domain: biomedical Document type: Study Language: en The following conclusions can be drawn from this study: (1) executive dysfunction significantly and positively predicts proactive and reactive aggression; (2) impulsivity partially mediates the relationship between executive dysfunction and proactive and reactive aggression; (3) in proactive aggression, impulsivity displays a significant predictive effect only among male adolescents; and (4), in reactive aggression, impulsivity has a significant predictive effect on both male and female adolescents, although the effect is more pronounced in males. | Study | biomedical | en | 0.999996 |
PMC11695427 | Section title: Introduction Educational score: 4.126720428466797 Domain: biomedical Document type: Review Language: en The number of older adults aged 65 and above is expected to nearly double from 52 million in 2018 to 95 million by 2060 in the United States . With aging, the likelihood for developing cognitive impairments increases, with an estimated 13.9 million individuals projected to be affected by Alzheimer’s disease and related dementias (ADRD) . Moreover, sensory loss, particularly hearing and vision impairment, has been linked to cognitive declines associated with aging and represent a significant issue among the aging population . It is estimated that approximately 25% of older adults will experience hearing loss ( Hearing loss and hearing aid use, n.d. ), 12% vision loss , and 11% both hearing and vision (dual sensory) loss. This figure rises to about 50% for those aged 75 and older. These sensory losses have significant negative impacts to quality of life; they can lead to social isolation , depression , and a higher risk of falls . Critically, declines in sensory/perceptual systems have also been shown to be predictive for developing ADRD , although the underlying mechanisms and causal relationships are still poorly understood. Thus, there is a great need for research that better characterizes relationships between sensory loss, age-related cognitive declines, and ADRD, as well as for clinical tools that can help with early detection, prevention, and appropriate management strategies to reduce the burden on the healthcare system and society at large. Section title: Introduction Educational score: 4.307373046875 Domain: biomedical Document type: Review Language: en Currently, the relationship between sensory loss and cognitive function is supported by two predominant theories: cascade theory and common cause theory . The cascade theory postulates that increased cognitive load due to impaired perceptual processes , adds strain to cognitive systems , leading to impaired performance. Further, there is a significant association of sensory loss with difficulty conducting complex tasks , increased social isolation , and reduced independence , which might further contribute to the development of ADRD . While the brain is capable of compensating for sensory losses to a certain degree, the associated neuroplasticity could become maladaptive resulting in structural and functional impairments, contributing to cognitive decline . Alternatively, the common cause theory assumes that both sensory and cognitive impairments are interconnected with atrophy and pathophysiological changes of the brain as a result of aging. Both theories explain the link between sensory deprivation and dementia risk, and they are often considered as overlapping concepts rather than mutually exclusive . Section title: Introduction Educational score: 3.9624106884002686 Domain: biomedical Document type: Review Language: en The interrelationship between sensory loss and cognitive decline in aging involves complex mechanisms that span biological, psychological, and social domains . Further, research suggests that several modifiable risk factors interact and impact cognitive aging which include high blood pressure, cardiovascular diseases, and diabetes. These individualized risk factors often occur already in middle age , suggesting that early prevention and intervention could have long-lasting impacts. Further, sociodemographic factors such as race/ethnicity, lifestyle, socioeconomic status, and educational attainment also impact ADRD risk . However, although numerous associations between sensory loss and ADRD have been demonstrated, causal relationships have yet to be unambiguously demonstrated , and the understanding of whether and how they interact with modifiable risk and sociodemographic factors is still limited . Section title: Introduction Educational score: 4.120454788208008 Domain: biomedical Document type: Review Language: en To better understand the relationships between sensory loss and cognitive function, there is a need for valid, reliable, and cost-effective measures that are sensitive enough to capture early sensory and cognitive declines. Furthermore, establishing causal relationships requires interventional and/or longitudinal data, which can be difficult to acquire due to extended timeframes. Yet, current research practices often fail to address central perceptual processes despite their clinical relevance. For example, assessments of low-level (e.g., basic sensory acuity tests) and mid-level (e.g., perceptual organization tasks) perceptual processing can provide fundamental information regarding people’s perceptual functions. While there are numerous tests of central perceptual processes, many of these are restricted to use in basic research and are rarely adopted for large-scale clinical use. There are multiple limitations of existing assessments of sensory and cognitive function that restrict translation to clinical practice and their implementation at scale. These include (1) requirement for specialized equipment and associated costs, (2) substantial time required for measurement, (3) lack of standardization, (4) lack of normative data on sensitivity and specificity of the different measures to predict cognitive decline and ADRD, and (5) lack of accessible assessment tools that include the latest advances in both theory and technology. In the following sections, we will review approaches to measure sensory/perceptual processes (with an emphasis on hearing and vision), and discuss opportunities to improve practice and implementation. Section title: Hearing and cognition Educational score: 4.307407855987549 Domain: biomedical Document type: Review Language: en Pure tone audiometry, used to establish the quietest sounds that a person can hear at different frequencies, constitutes the gold-standard clinical assessment of hearing . Elevated hearing thresholds captured by these clinical audiograms are the traditional definition of hearing loss, affecting 20% of people worldwide . They are thought to represent declines in the peripheral auditory system. The risk for this peripheral hearing loss increases with age , with over 60% of all individuals with hearing impairment being older than 50, and over 50% of people experiencing at least a moderate level of peripheral hearing loss by the age of 90 . Meta-analytic work using large-scale longitudinal studies investigating the relationship between peripheral hearing loss and cognitive decline has identified peripheral hearing loss in mid-life as a major modifiable risk factor, predicting the development of dementia later in life . Importantly, peripheral hearing loss can be detected years before the clinical manifestation of dementia , suggesting that peripheral hearing loss could serve both, as a critical early marker and a target for intervention . Section title: Hearing and cognition Educational score: 4.489139080047607 Domain: biomedical Document type: Review Language: en Although pure tone audiometry is standardized and has clear clinical relevance, there is a high prevalence of individuals with relatively normal audiograms, but who experience and self-report great hearing difficulties, in particular, understanding speech in noisy conditions, such as following a conversation in a restaurant . Furthermore, clinical categories such as hidden hearing loss or central auditory processing disorder are well-established and illustrate that hearing difficulty can be only partially assessed with pure tone detection measures. Contributions to hidden hearing loss are manifold where a deficit or disconnection in any part of the ascending auditory pathways, from the cochlea to the brainstem and midbrain to the auditory cortices and onward to frontal and parietal regions, can impair auditory processing . Central auditory processing functional deficits associated with age [e.g., central presbycusis ] can include difficulties in processing rapidly changing sounds , locating sounds in space , attending to sounds of interest , processing of contextual cues that guide meaning , and understanding speech in noisy conditions . To successfully understand speech, we need to detect and discriminate rapid changes in the temporal and spectral structures of broad-band sounds in complex auditory scenes , where speech signal needs to be segregated from the competition to be successfully perceived . In addition, cognitive processes including attention and working memory also contribute to speech-in-noise comprehension . Section title: Hearing and cognition Educational score: 3.986476182937622 Domain: biomedical Document type: Study Language: en While complaints of not being able to hear conversational partners are ubiquitous among older adults , assessments of central auditory processes are not broadly administered, despite the fact that self-reported hearing difficulties may be better explained by speech-in-competition measures than the audiogram . Further, there is growing evidence that central auditory processes may be predictors of ADRD, but that relationship remains largely understudied. For example, Gates et al. and Mohammed et al. found that the Dichotic Sentence Identification test, a standard measure of speech-in-competition, was associated with elevated risk for dementia (up to 4 times), even after controlling for pure tone audiogram thresholds. However, there are no gold-standard tests to measure self-reported hearing ability, speech-in-competition, or other central auditory processes. Thus, there is a substantial need to establish and standardize measures of hearing to accurately measure possible dysfunction at different stages of auditory processing and establish their relationships to age-related cognitive decline and ADRD. Section title: Vision and cognition Educational score: 3.067929983139038 Domain: biomedical Document type: Other Language: en Measures of visual acuity, used to determine the smallest characters a person can read, currently serve as the gold standard of assessing visual abilities. The Snellen chart, introduced in 1862, is the earliest and best-known standardized visual acuity measurement tool, It consists of letters arranged in rows of decreasing size . Section title: Vision and cognition Educational score: 4.414825439453125 Domain: biomedical Document type: Review Language: en Visual impairment, as captured with low-level measures such as visual acuity, can be caused by several factors, including altered visual inputs of optic or retinal nature, or impairment of cortical perceptual processing. For example, in macular degeneration - the most common cause of visual impairment in the western world - degeneration of foveal photoreceptors leads to central vision loss. This conditions greatly compromise everyday tasks such as reading, navigating, and recognizing faces. However, losing central vision deprives patients not only of their retinal location with the highest resolution, but also it impacts their oculomotor attentional processes . The relationship of the visual system with higher level processing regions is reflected in the tight connection between visual and cognitive impairment, with multiple studies showing that macular degeneration and Alzheimer’s disease share common risk factors and histopathological changes. Furthermore, there is epidemiological evidence linking macular degeneration to cognitive impairment including impaired performance in tasks of memory, executive functions, and global cognition . Section title: Vision and cognition Educational score: 4.22581148147583 Domain: biomedical Document type: Review Language: en Studies that provide causal evidence for the relationship between vision loss and ADRD are at its infancy. In regard to the common cause model, tests using degraded visual letters show promise to detect visual Alzheimer’s disease . In regard to the cascade theory, recent investigations have demonstrated that cataract surgery, an extremely common visual restoration procedure, is associated with amelioration of age-related cognitive declines . Similarly, in glaucoma, a neurodegenerative condition affecting both visual and cognitive functions, strategies such as vision rehabilitation (i.e., visual and cognitive training) , assistive technologies (screen readers or text-to-speech software) , and pharmacological drugs (e.g., eye drops) have shown potential benefits on improving cognitive performance, suggesting that visual impairment could be a modifiable risk factor for pathological cognitive decline. Although visual acuity is an important measure of vision function, it is only one of many different visual processes that can be relevant to understand visual impairments. As with hearing, there are numerous stages of visual processing that are all important to functional vision. For example, our ability to read words or recognize faces, depends on our ability to process small changes in luminance contrast, relative depths, and motion, all of which help us detect important visual features that we integrate into objects and then process these using higher cognitive functions including attention and memory. Visual impairments related to higher-level visual functions are linked to reduced quality of life and negative psychological well-being. Section title: Dual sensory loss Educational score: 3.9271647930145264 Domain: biomedical Document type: Study Language: en While it is common for research on sensory impairments to focus on single conditions, dual sensory loss refers to cases where impairments impact multiple senses. For example, Desai and colleagues conducted an epidemiological study that suggested that at least 11% of the population experiences dual hearing and vision loss, and most people express concerns of both hearing and vision as they age . Furthermore, there is evidence that the impacts of dual sensory loss accelerates the risk of developing ADRD, compared to those without sensory impairments . In line with this, other work has shown that people with dual sensory loss are twice as likely to develop ADRD than those without any sensory impairments . Section title: Dual sensory loss Educational score: 3.983217239379883 Domain: biomedical Document type: Study Language: en However, dual sensory loss often remains undetected due to fragmented healthcare systems (i.e., the division of audiology and optometry) and the prioritization of more acute medical conditions. Thus, in addition to the need to better characterize our individual senses, it is also important to measure how they work together. For example, tests of audiovisual integration , can help us understand how sensory information from multiple modalities interact. In addition, tests like the audio-visual divided attention task, where attention must select between, or be divided among sensory streams can help us understand better how sensory systems can also compete for attentional and memory resources . Further, it is important to understand relationships with sense beyond just hearing and vision. For example, vestibular functions that have strong impacts on mobility processes such as balance control impairments and vestibular dysfunction leading to falls, also interact with both, hearing and vision, as well as higher cognitive functions, and ultimately, predict ADRD . Section title: Recommendations Educational score: 3.890820264816284 Domain: biomedical Document type: Review Language: en As highlighted in the sections above, sensory/perceptual loss is a multidimensional set of conditions that has complex, and likely insufficiently understood relationships with cognition and ADRD. Our current medical system has traditionally separated clinical approaches to treat hearing loss, vision loss, as well as evaluations and treatments for motor and cognitive function, which has systematically limited the research required to understand how co-occurring declines across all these systems interact with aging (another separate medical discipline), and ADRD. While it is obvious to all of us that with age, our vision, hearing, mobility, as well as cognition decline, however, a better understanding of how these systems interact to impact our long-term health and wellbeing requires a paradigm shift and creation of new standards and practices. Section title: Recommendations Educational score: 4.045854091644287 Domain: biomedical Document type: Study Language: en For example, while existing standards such as pure tone audiometry for hearing and acuity tests for vision do provide useful functional evaluations that lead to easily deployable interventions such hearing aids and vision aids (e.g., glasses, contacts), these assessments fall short in capturing the complex, real-world sensory impairments and cognitive interactions that affect everyday life. A tangible example would involve engaging in a simple conversation with friends at a restaurant that involves complex interactions between central auditory processing to select and processing the voices of our conversational partners over the many distracting noise sources. These processes are facilitated by our visual system that can view our friends’ lips, and multisensory processing to integrate this information to understand their speech. Lastly, encouraging simultaneous performance of more than one task (cognitive-motor, motor-motor, cognitive-cognitive) as we process other relevant visual information related to our food and drinks, and coordinate with our motor system to bring food to our mouth. All these examples further tap into cognitive resources involving attention, memory and cognitive control processes related to speech comprehension. Section title: Recommendations Educational score: 3.9294090270996094 Domain: biomedical Document type: Review Language: en To address the complex, multiple domain activities that are ubiquitous of daily functions, we need new paradigms that better address the dimensionality and complexity of sensory/perceptual and cognitive processes that people rely upon, and to understand the individual trajectories of these, and their interactions with aging processes. These can be facilitated by advancements in consumer technology, peoples’ phones and computers, and even more impressively, new extended reality (XR) systems that have more advanced audio and visual processing systems that are found in many high-end clinical evaluation systems. This gives rise to transformative potential to advance both in-patient and out-patient assessments of sensory/perceptual and cognitive processes. Further, with gyroscopes and accelerometers becoming increasingly standard in smart-phone, smart watches, or XR headsets, it could be convenient to evaluate postural control in simple single as well as dual tasking conditions that can help understand dual models of cognitive decline among others. Section title: Recommendations Educational score: 4.329031944274902 Domain: biomedical Document type: Review Language: en Building on these technological advancements highlights the transformative potential of low-cost consumer technologies for home-based clinical screening . For example, it is easy for someone to conduct a large battery of tests of both peripheral and central processing with a high degree of fidelity. In the case of hearing, tests of spatial release from masking , where one must listen to the instructions of one speaker while ignoring other people speaking similar but misleading instructions, can provide a measure of hearing in complex, real-world-like environments. Digital technology also allows for convenient testing of hearing handicap, pure-tone sensitivity, spectral, temporal, and spectral-temporal processing, and other speech in competition tests in at-home settings , and thus, improving the tests’ real-world relevance. Similarly, in the case of vision, complex tasks such as trail making or visual search can be digitally administered and followed-up by tests of more basic visual functions such acuity, contrast and color sensitivity, stereo vision, and motion processing . These measures can be complemented with standard neuropsychological tasks capturing memory, executive functioning, language, and visuospatial reasoning, and at the same time, their digital administration can reveal data that go beyond traditional paper-pencil tests that have dominated standard cognitive evaluation. Furthermore, the integration of motion sensors in these devices facilitates the estimation of postural control, which is crucial for understanding cognitive decline through dual-tasking conditions and dual sensory loss . To fully leverage these advancements however, the development of normative data and standardized protocols is essential, ensuring accuracy and reliability across various settings . Utilizing widely available and low-cost consumer devices also improves accessibility and efficiency, allowing for quicker and more frequent evaluations, which is particularly beneficial for early detection, monitoring progressive conditions, and adjusting treatments promptly . As such, digital assessments can provide a unique opportunity to advance our understanding of the multidimensional relationships between sensory/perceptual functions and cognition in ADRD, however, it will be important to address key challenges such as standardization, time efficiency, and accessibility. | Review | biomedical | en | 0.999994 |
PMC11695431 | Section title: Introduction Educational score: 4.50554084777832 Domain: biomedical Document type: Review Language: en Synovial Sarcoma (SS) is a highly aggressive malignant tumor derived from undifferentiated mesenchymal cells, which accounts for 5%–10% of malignant tumors of mesenchymal tissue ( 1 ). Synovial sarcoma can occur in all age groups, but predominantly in young adults. The age of patients is generally between 20 and 40 years old, and there is no significant difference between men and women. It can be found in various parts of the body, preferably in the vicinity of large joints, and is closely related to tendons, tendon sheaths, synovial bursae, joint capsules. It can also occur in parts without synovium, and usually does not invade the joint itself ( 2 ). SS is not differentiated from synovium but consists of spindle cells of unknown origin and/or nests of epithelioid cells of varying degrees of differentiation. According to the presence of both types of cells, SS could be divided into monophadiform and biphasic types, or an undifferentiated type containing only poorly differentiated fibrous spindle cells ( 2 ). No known risk factors for synovial sarcoma have been identified, but the disease is associated with the t (X; 18) (p11; q11) chromosomal translocation. SS18(SYT)-SSX fusion gene testing is the gold standard for the diagnosis of SS, while immunohistochemistry and imaging tests are used as auxiliary diagnostic tools ( 3 ). SS has a poor prognosis, with reported 5-year survival rates of 36%–76% and 10-year survival rates of 20%–63%. More than 50% of patients recurring within 2 years, while about 40% of patients will metastasise to the lung, bone, and other sites ( 4 ). Surgery supplemented by postoperative radiotherapy/chemotherapy is considered the mainstay of treatment. Section title: Introduction Educational score: 2.9665098190307617 Domain: clinical Document type: Clinical case Language: en We report on a 16-year-old Chinese female diagnosed with synovial sarcoma who underwent two surgical treatments and regular chemotherapy. Follow-up did not reveal any recurrence of the original disease, but bilateral lung metastases were detected. We also reviewed the relevant literature on synovial sarcoma of the thigh over a 2 decades period. Section title: Case presentation Educational score: 1.9175691604614258 Domain: clinical Document type: Clinical case Language: en A 20-year-old female presented to the hospital with a history of post-exercise pain for more than 11 months. She found a mass on her right thigh that was painful and swollen after compression. Section title: Case presentation Educational score: 2.452678680419922 Domain: clinical Document type: Clinical case Language: en Physical examination: palpable mass on the right medial thigh was poorly demarcated from the surrounding tissues on palpation, with pressure pain accompanied by swelling, and the skin around the mass was normal in color and inactive. Section title: Case presentation Educational score: 3.968579053878784 Domain: clinical Document type: Clinical case Language: en Ultrasound: A slightly hypoechoic mass measuring about 7.2 × 5.6 × 6.4 cm was found in the medialis longus muscle of the lower middle thigh on the right side, with an irregular shape, poorly defined borders, and poorly homogeneous internal echogenicity, and the peripheral superficial femoral vein and saphenous vein of the mass were poorly displayed , and color Doppler showed that rich blood flow signals could be seen in the mass . Ultrasound suggests that further examination is recommended. MRI:Soft tissue mass in the medial muscle of the middle right thigh (short and long adductors in the center, and part of the medial femoral muscle), foliated, with slightly high signal in T1WI, high signal in T2WI, and widely diffusion-restricted in DWI sequences; the mass was clearly delimited from the right femur, with intact cortex, and no abnormal signal foci were seen in the bone marrow cavity . The MRI shows a high likelihood of malignancy and an enhanced scan is recommended. X-ray radiography showed irregular and massive tumor vascular shadow in the soft tissue area of the right mid-femoral femur, which was supplied by the branch of right superficial femoral artery and deep femoral artery, and dense tumor staining was seen in the parenchymal stage, and there was no obvious venous early appearance . X-ray radiography showed that a soft tissue malignant tumor was considered in the middle of the right femur. Chest CT showed no significant metastases in both lungs . Section title: Case presentation Educational score: 4.0274786949157715 Domain: clinical Document type: Clinical case Language: en The patient underwent surgery. The operation showed that the tumor was wrapped in the sarcolemma, without complete capsule, and was friable, partially fish-like, wrapping the femoral artery and saphenous nerve. The boundary between the tumor and the surrounding tissue was not clear, and it extended to the deep tissue. Because the tumor was too large and poorly demarcated from the surrounding tissues to be completely resected, local tumor tissue was excised and sent for pathological examination. Microscopic manifestation: the tumor cells are densely proliferated, with flaky, nested mass, striated distribution, infiltrative growth, the nuclei are ovoid, fat pike-shaped, with visible nucleoli, and the nuclear schizophrenia is easy to be seen . Immunohistochemistry: CD99 (partially+), Bcl-2 (partially+), EMA (mostly+), S100 (−), Desmin (−). Molecular pathology:SS18 gene translocation testing showed that 55 cells exhibited separated red and green signals among 100 counted cells, accounting for 55%. The reference value for a positive result is >15%. Therefore, the result is considered positive, indicating the presence of SS18 gene translocation . The combination of immunohistochemical and molecular test results was consistent with synovial sarcoma. Two postoperative chemotherapy sessions were performed, with a chemotherapy regimen of biliverdin, cisplatin, and isocyclophosphamide. The second operation was performed 2 months after the operation. The operation showed that the tumor was solid, located below the deep fascia, the tumor was large with unclear borders, and the proximal part of the tumor was obviously adherent to the saphenous vein and femoral artery, encircling the vascular bundles and femoral nerve. The tumor invades the suture muscle superficially, the medial retractor muscle group deeply, and part of the medial femoral muscle distally. Regular chemotherapy was administered postoperatively, and the treatment regimen was liposomal isocyclophosphamide and doxorubicin. Section title: Case presentation Educational score: 1.4848304986953735 Domain: clinical Document type: Clinical case Language: en Postoperative follow-up of the patient showed no definite recurrence in the right lower extremity.13 months after surgery, chest CT showed multiple metastases in both lungs . The patient is now on regular chemotherapy with routine follow-up. Section title: Discussion Educational score: 4.019641876220703 Domain: biomedical Document type: Study Language: en Synovial sarcoma is a rare, malignant, highly graded soft tissue tumor. The age-adjusted incidence of this subtype is 00.81 per 1 million children and 1.42 per 1 million adults ( 5 ). We searched the literature for the past 2 decades and found a total of 25 reports of cases of synovial sarcoma of the thigh ( 1 , 6 – 29 ) ( Table 1 ). These cases involved patients ranging in age from 13 to 75 years with a mean age of 38.2 years. There were 14 male patients (56.0%) and 11 female patients (44.0%). A total of 25 lesions were involved in these 25 reports in the literature, 14 of which were located on the left side and 11 on the right side. The size of the lesions was documented in 20 reports showing that the maximum diameter of all lesions was more than 3 cm, and the maximum diameter of 10 lesions was more than 10 cm, accounting for 50%. In our patient, the maximum diameter of the lesion was 7.2 cm. These reports indicated that the size of the lesions was generally large, which may be related to the fact that the clinical manifestations of the disease were not obvious in the early stage and did not attract much attention from the patients. As a result, by the time the patient is seen, the lesion is already relatively large. Section title: Discussion Educational score: 4.038153171539307 Domain: biomedical Document type: Review Language: en Of this literature, only 6 explicitly reported recurrence of the original disease. Among them, the recurrence occurred in 2 cases, and the other 4 cases recurrence did not occur. Distant metastasis was explicitly reported in 17 articles. Of these, 11 patients developed metastases, while the other 6 did not. Of the cases in which metastasis occurred, 6 metastasized to lung tissue, 2 to the pancreas, and one each to the chest wall, endocardium, adrenal glands, stomach wall, and lumbar spine. Nine reports clearly documented the deaths of patients. Of these reports, three patients died within 5 years of lesion detection, whereas the other six patients did not experience a fatal event during the follow-up period. In the literature, postoperative follow-up for patients with SS primarily relies on imaging examinations, focusing on chest CT, abdominal ultrasound, bilateral lower limb MRI, and PET-CT to detect subtle metastatic lesions that may be challenging to identify with different imaging modalities. Section title: Discussion Educational score: 3.998840570449829 Domain: biomedical Document type: Study Language: en Out of these 25 case reports, only 1 paper mentioned that the lesion was supplied by the deep femoral artery. In this case, radiographic examination showed that the blood supply to the lesion came from branches of the superficial femoral artery and the deep femoral artery. In 11 of these reports, the tissues involved in the lesion were described, including the sciatic nerve in 4 cases, the femoral nerve in 1 case, the femoral vein in 2 cases, the superficial femoral artery in 1 case, the superficial femoral vein in 1 case, the biceps femoris muscle in 2 cases, the rectus femoris muscle in 1 case, the middle femoral muscle in 1 case, the adductor femoris muscle in 1 case, and the adductor longus muscle in 1 case. For this case, we concluded from the imaging presentation and intraoperative situation that the lesion invaded several arteries, such as the right superficial femoral artery and the deep femoral artery, and veins, such as the superficial femoral vein and the great saphenous vein. There was also invasion of the femoral nerve, as well as several muscle tissues, including the adductor brevis, adductor longus, part of vastus medialis and sartorius. Section title: Discussion Educational score: 4.09425163269043 Domain: biomedical Document type: Study Language: en Imaging examination plays an important role in the localization and characterization of the disease, and provides guidance for the treatment of the disease. Of these 25 case reports, only 4 described the ultrasonographic appearance of the lesion ( Table 2 ). These reports showed a mixed echogenic mass of the lesion, in which calcification was seen in 1 case, areas of echo-less liquefaction were 2 in two cases, and a small amount of blood flow signal was seen in the lesion on color Doppler. In contrast, the ultrasound presentation of the lesion in this case was a slightly hypoechoic mass, with no clear areas of calcification or liquefaction observed. In addition, the lesion and the adjacent superficial femoral vein and great saphenous vein were not clearly visualized, while the blood flow signal in the lesion was slightly rich on color Doppler. Based on this case, we can summarize that the SS is a solid hypoechoic mass located near the joint, with variable size, well-defined borders, and no invasion of adjacent joints. The smaller masses have relatively regular shapes and homogeneous internal echogenicity, while the larger ones tend to have irregular shapes and heterogeneous internal echogenicity, with possible occurrences of liquefaction and calcification. Color Doppler imaging reveals abundant blood flow signals within the mass, exhibiting a patchy or branching distribution. Section title: Discussion Educational score: 4.07595157623291 Domain: biomedical Document type: Review Language: en Ultrasound can demonstrate the extent of involvement of surrounding muscles and blood vessels, as well as the vascular supply; however, a definitive diagnosis of SS still requires differentiation from other diseases, such as malignant peripheral nerve sheath tumor, malignant fibrous histiocytoma, invasive fibroma, etc. Malignant peripheral nerve sheath tumors are commonly found in the thigh, buttock, upper arm, and paravertebral region. They typically present as heterogeneous hypoechoic masses, which may show irregular, varying thickness strands within. Cystic changes may be observed inside, and the margins may exhibit a capsule-like strong echogenicity. Malignant fibrous histiocytoma typically runs parallel to the long axis of the body and is located within the muscle layer or subcutaneously, which is a key distinguishing feature. Invasive fibroma presents as a hypoechoic mass with interspersed areas of hyperechogenicity. Expansile growths appear as rounded or oval shapes, with some showing a pseudocapsule. Section title: Discussion Educational score: 4.098360061645508 Domain: biomedical Document type: Study Language: en MRI has good soft tissue resolution, especially in the nervous system and bone and joint systems. Of these 25 case reports, 13 described the MRI manifestations of the lesions ( Table 2 ). MRI showed the lesion to be a soft tissue signal with iso-or hypointensity on T1-weighted images and mixed high and low signal on T2-weighted images, and enhancement scans showed the lesion to show inhomogeneous enhancement. Only one of these reports described a lesion with invasion of adjacent muscles and encircling the femoral artery. “Triple-sign” is always mentioned in the literature ( 30 ), that is, synovial sarcoma has three signal areas of high, medium and low signal in T2-weighted images. In this case, MRI showed isointense and hyperintense T2-weighted images of the lesions, hyperintense signals of cystic degeneration and small pieces of necrosis, and hypointense signals of calcification, fibrous components and old hemorrhage. The lesion invaded the adductor brevis, adductor longus and part of the adductor femoris, but did not invade the right femur. The lesion wrapped around the right superficial femoral artery, indicating the difficulty of surgery. When the fascia of the muscle tissue surrounding the lesion is unclear and there is a disruption in the normal alignment of the muscle fibers, the possibility of invasion should be considered. When the lesion encases a blood vessel or is located adjacent to one, with the blood vessel appearing tortuous and enlarged, the possibility of invasion should be considered. Section title: Discussion Educational score: 4.120736598968506 Domain: biomedical Document type: Review Language: en CT is not the preferred imaging modality for soft tissue lesions, as evidenced by the reported cases and this case, which did not include CT scans of the primary lesion. However, the study by Mickael et al. indicates that CT is more sensitive to calcific components within the lesion ( 30 ). The presence of calcification in SS lesions is associated with better overall survival, and CT imaging can be helpful in predicting the prognosis of SS patients. Ultrasound examination can partially reveal the presence of hemorrhage, necrosis, and calcification within SS lesions, show whether the lesion has a capsule, and provide a more visual assessment of blood flow in the lesion’s center, as well as its proximity and relationship with surrounding blood vessels. MRI can perform multiparametric high-contrast imaging of SS lesions, providing more comprehensive diagnostic information. This enhances the ability to characterize the lesions in detail, assess their extent, and evaluate their relationship with adjacent structures. However, MRI is not sensitive to the detection of calcification within lesions and the surrounding cortical bone. This limitation can affect the assessment of certain characteristics of the lesions. CT and PET scans are better suited for detecting distant metastases, providing valuable information about the presence and extent of metastatic disease. Therefore, the diagnosis of SS should integrate multiple imaging modalities to obtain a comprehensive assessment of the lesions and their characteristics. Section title: Discussion Educational score: 4.220752239227295 Domain: biomedical Document type: Study Language: en Among the 25 case reports, 15 mentioned pathological classification, of which 9 cases were monophasic type, 5 cases were biphasic type, and only 1 case was undifferentiated type. In view of this case, the microscopic observation showed that the epithelioid tumor cells and spindle tumor cells were mixed and densely proliferated, presenting nested clusters and striated distributions, with characteristics of infiltrative growth, which was in line with the manifestation of biphasic tumors. SS18 gene translocation was detected in 9 cases. Immunohistochemical testing was performed in 13 cases, of which 5 were positive for Bcl-2 antibodies and 7 were positive for EMA. For this patient, immunohistochemistry results showed positive Bcl-2 antibodies and EMA, and these results helped to confirm the diagnosis of SS. Section title: Conclusion Educational score: 3.858924627304077 Domain: biomedical Document type: Clinical case Language: en In conclusion, synovial sarcoma is a rare malignant mesenchymal tumor, so its accurate diagnosis is essential for treatment and prognosis. We report the diagnosis of biphasic synovial sarcoma in a 16-year-old Chinese female, and we provide a comprehensive and detailed description of her clinical symptoms, imaging manifestations, surgery, and pathologic findings. Synovial sarcoma in the imaging can show certain characteristics, but its specificity is low, need to identify with a variety of other soft tissue tumors. Histopathological and immunohistochemical findings are still needed to make a definitive diagnosis. | Review | biomedical | en | 0.999995 |
PMC11695432 | Section title: Introduction Educational score: 2.582932233810425 Domain: other Document type: Study Language: en The rapid proliferation of the internet, computers, smartphones, and electronic devices over recent decades has led to an increased risk of problematic internet use (PIU), a public health concern recognized across many countries by the World Health Organization ( 1 ). The COVID-19 pandemic has exacerbated this issue due to the necessity of prolonged physical isolation ( 2 ). To compensate for the lack of face-to-face social interactions, and the psychological needs unmet in their real lives, individuals increasingly rely on the internet ( 3–5 ), thereby heightening the risk of PIU ( 6 ). Numerous studies have investigated excessive internet use among adolescent students during the pandemic, revealing a worsening trend of PIU within this demographic ( 7–9 ). Section title: Introduction Educational score: 1.723034143447876 Domain: other Document type: Review Language: en PIU, an umbrella term replacing earlier terms such as internet addiction ( 10 ), internet dependence ( 11 ), and compulsive internet use ( 12 ), refers to a maladaptive pattern of internet use characterized by loss of control, negative consequences, and compulsive thoughts about internet access ( 13 , 14 ). Current research on PIU includes the following two primary aspects: Firstly, studies have documented the prevalence of PIU across diverse populations as seen in Guo et al., Zhao et al., and Mohler-Kuo et al. ( 15–17 ). However, these studies have shown inconsistent results, according to Burkauskas et al. ( 18 ). This inconsistency in prevalence rates across studies complicates the understanding of PIU’s scope and impact. Section title: Introduction Educational score: 3.8664705753326416 Domain: biomedical Document type: Study Language: en Secondly, the relationship between PIU and other factors, particularly mental health, has received significant attention from researchers ( 19–21 ), with a general consensus on the negative correlation between PIU and individual mental health. Research indicates that PIU is associated with emotional disorders ( 22–24 ) and is positively correlated with stress, anxiety, and depression among university students ( 25–28 ), as well as loneliness ( 29 , 30 ), low self-esteem ( 31 , 32 ), and stigmatization ( 33 ). Devine et al. ( 34 ) have underscored the critical need to gather evidence of PIU from diverse individuals to achieve a comprehensive understanding of the issue. These findings highlight the importance of further research to understand the nuances of PIU and its impact on mental health across different demographic groups. Section title: Introduction Educational score: 3.8391635417938232 Domain: biomedical Document type: Study Language: en More specifically, the issue of PIU during the COVID-19 pandemic was suspected to be exacerbated due to the increased time spent online ( 3 , 5 , 18 ). The existing literature on PIU during the pandemic reveals several notable gaps that warrant further investigation. First, the prevalence estimates of PIU during the pandemic are highly inconsistent across studies ( 18 ). For example, Zhao et al. ( 16 ) conducted a cross-sectional study among 11,254 Chinese university students and found a PIU prevalence rate of 28.4%. In contrast, two Hungarian studies ( 35 , 36 ) reported PIU prevalence rates of 3.9% (485 hospital staff members) and 5.2% (1,817 high school teachers). A Swiss study by Mohler-Kuo et al. ( 17 ) which included 1,146 children and adolescents and 1,627 young adults, estimated the PIU prevalence rate to be 30.1% for children and adolescents and 21.3% for young adults. Given this inconsistency in PIU prevalence rates, it is essential to conduct more research to better understand this issue. Section title: Introduction Educational score: 3.5161936283111572 Domain: biomedical Document type: Study Language: en Second, the majority of research on PIU has been concentrated on younger demographics, such as adolescents and university students ( 21 , 22 , 25 , 26 ), leaving a gap in the research on PIU among older adults (40+ years) ( 37 ). This raises particular concerns regarding the increasing longevity and growing proportion of adults in the population ( 38 ). Empirical studies have not only identified the presence of PIU within this demographic ( 34 , 38 , 39 ), but also highlighted its potential to increase health risks, including greater social isolation, depression, and increased suicide rates ( 37 , 39 ). Given the importance of improving the quality of life for older adults for familial wellbeing and social stability, especially in the context of an aging population ( 40 , 41 ), it is imperative to examine the prevalence, causes, and consequences of PIU among older adults, with a particular focus on older teachers within the education system, who play a significant role and are interconnected with the student population. Section title: Introduction Educational score: 1.1718908548355103 Domain: other Document type: Study Language: en Within the education system, teachers play a crucial role and have been identified as a group that experiences significant stress ( 42–44 ), especially during the COVID-19 pandemic. The pandemic led to the global closure of schools ( 45 ), significantly impacting the education sector and the teacher community ( 46 ). Teachers were confronted with the mandatory shift to online teaching ( 47 ) and faced technical and logistical challenges as they transitioned from classroom to home-based instruction ( 48 ), along with increased workloads and diminished communication with students and parents ( 49 ). Consequently, the literature has shown that COVID-19 has inflicted considerable psychological distress and excessive internet use on teachers ( 50 , 51 ). Some studies explored the impact of teachers’ PIU on their mental health ( 44 , 52 , 53 ). Section title: Introduction Educational score: 1.1810060739517212 Domain: other Document type: Other Language: en Addressing the prevalence and impact of PIU among teachers is essential, as their psychological wellbeing greatly affects teaching effectiveness ( 54 ). Previous research has shown that teachers’ mental health is closely related to student behavior, potentially leading to worse academic achievement and reduced student motivation ( 55 , 56 ). While these studies have shed light on the impact of teachers’ PIU on their mental health, a significant gap remains in the literature, as there has been no specific focus on older teachers. This oversight is a critical area for future research, as understanding the challenges faced by older teachers can inform targeted interventions and support strategies to promote healthy internet use and overall wellbeing. Section title: Introduction Educational score: 3.909907579421997 Domain: biomedical Document type: Study Language: en Third, most current studies report the prevalence of PIU among participants using a single cross-sectional design. While this approach captures the relationship between PIU and individual psychological distress at a specific moment, it falls short of providing a comprehensive analysis. A more robust methodology would be to employ multiple cross-sectional studies (called repeated cross-sectional studies) conducted over time, which would offer a dynamic view of how PIU prevalence has fluctuated among target groups and how it has interacted with psychological distress throughout different phases of the COVID-19 pandemic. Section title: Introduction Educational score: 3.927020311355591 Domain: biomedical Document type: Study Language: en In a prior study, Soriano et al. utilized a repeated cross-sectional survey to study the prevalence of chronic obstructive pulmonary disease (COPD) in Spain using two different samples at two distinct points in time: 1997 and 2007 ( 57 ). Similarly, to ascertain the prevalence of PIU among older school teachers during the 3 years of the COVID-19 pandemic, we conducted a repeated cross-sectional survey at three different periods. This methodology allows researchers to gain a deeper understanding of how the population engaging in PIU has changed over time and the association between PIU and mental health outcomes as the pandemic progressed and societal circumstances changed. Section title: Introduction Educational score: 2.5767099857330322 Domain: other Document type: Study Language: en To address these gaps, this study focuses on older teachers in China, conducting three cross-sectional studies to evaluate their prevalence of PIU across 3 years of the COVID-19 pandemic and examining the impact of PIU on psychological distress. The first study, set in the immediate aftermath of the pandemic outbreak , explores the influence of pandemic fear on PIU and psychological distress among older teachers. This study aims to understand how the initial shock and uncertainty of the pandemic affected teachers’ problematic online behaviors and mental wellbeing. The second study, conducted during the mid-pandemic period of online teaching in schools , investigates the impact of PIU on teachers’ psychological need thwarting (PNT) related to online teaching and distress. This study examines how the shift to remote education and the increased reliance on technology may have exacerbated PIU and its negative consequences for teachers’ psychological needs and wellbeing. The third study, carried out during the late-pandemic transition back to offline teaching , examines the effects of PIU on occupational burnout and psychological distress among older teachers. This study aims to understand how the lingering effects of PIU, developed during the pandemic, may continue to impact teachers’ professional and mental health as they readjust to in-person teaching. Section title: Introduction Educational score: 1.9694105386734009 Domain: other Document type: Study Language: en In this study, we employ problematic smartphone use (PSU) as an indicator of PIU among older teachers. This decision is based on the widespread use of smartphones in China, with 99.9% of Chinese internet users accessing the internet via mobile phones, and older individuals (aged 40 and above) accounting for a significant 48.5% of this population. Moreover, there is a significant overlap between PSU and PIU ( 58 ), and a growing body of research demonstrates the close relationship between PSU and mental health ( 59 , 60 ). Section title: Introduction Educational score: 1.6548562049865723 Domain: other Document type: Study Language: en By conducting these three sub-studies, this research provides a comprehensive analysis of PIU and its effects on older teachers’ psychological distress throughout the different stages of the COVID-19 pandemic. The following sections will detail the research hypotheses for each sub-study. Section title: Research hypothesis Educational score: 2.1511354446411133 Domain: biomedical Document type: Study Language: en Three conceptual models were tested in three distinct studies, and their respective hypotheses are depicted in Figures 1 – 3 . Section title: Hypotheses for study 1: the effect of fear of COVID-19 on problematic internet use, and psychological distress Educational score: 2.3744635581970215 Domain: biomedical Document type: Study Language: en The COVID-19 pandemic has led to widespread fear and the implementation of preventive measures like physical distancing. As a result, people have increasingly turned to social networks for medical information, stress relief, and psychological comfort, potentially leading to PIU ( 61 , 62 ). Teachers face additional challenges that may exacerbate PIU, such as the need to use social networks for online teaching and communication with students and parents ( 48 ). Thus, we hypothesize that fear of COVID-19 is significantly and positively correlated with PIU among older teachers. Section title: Hypotheses for study 1: the effect of fear of COVID-19 on problematic internet use, and psychological distress Educational score: 2.4600188732147217 Domain: biomedical Document type: Study Language: en The widespread fear caused by the COVID-19 pandemic has not only been associated with increased PIU but also with various forms of psychological distress, including depression, anxiety, and post-traumatic stress ( 44 , 63–65 ). Specifically, Yi et al. ( 44 ) found that Chinese primary and middle school teachers’ fear of COVID-19 was closely related to their psychological distress. As older teachers are a subset of this group, we hypothesize that their fear of COVID-19 is positively correlated with psychological distress. Section title: Hypotheses for study 1: the effect of fear of COVID-19 on problematic internet use, and psychological distress Educational score: 1.5692178010940552 Domain: other Document type: Study Language: en Moreover, according to the Resource Model of Self-Control ( 66 ), PIU consumes a significant amount of an individual’s limited self-control resources ( 67 ). For older teachers, who have relatively less energy, this may lead to ego depletion ( 66 ) and ultimately result in psychological distress ( 68 ). Research has shown that PIU is significantly associated with the psychological wellbeing of primary and middle school teachers ( 44 , 52 , 53 ). Therefore, we hypothesize that PIU is positively correlated with psychological distress among older teachers. Section title: Hypotheses for study 1: the effect of fear of COVID-19 on problematic internet use, and psychological distress Educational score: 2.498992681503296 Domain: biomedical Document type: Study Language: en Considering the relationships between fear of COVID-19, PIU, and psychological distress, we speculate that PIU may have a mediating effect between fear of COVID-19 and psychological distress. In other words, fear of COVID-19 may influence psychological distress through increased PIU. Thus, we hypothesize that PIU mediates the relationship between fear of COVID-19 and psychological distress. Section title: Hypotheses for study 2: the effect of problematic internet use on psychological need thwarting of online teaching and psychological distress Educational score: 1.2214181423187256 Domain: other Document type: Other Language: en Drawing from the Resource Model of Self-Control, PIU leads teachers to invest significant time and energy in online activities, consuming their self-control resources and resulting in ego depletion ( 66 ), which in turn affects their psychological distress (refer to the above hypothesis in study 1). Section title: Hypotheses for study 2: the effect of problematic internet use on psychological need thwarting of online teaching and psychological distress Educational score: 1.1556205749511719 Domain: other Document type: Other Language: en Moreover, during the COVID-19 pandemic, teachers have faced numerous challenges in adapting to online teaching, which has led to increased stress and workload ( 44 , 68 ). When teachers engage in PIU, it can further exacerbate these challenges by taking time and attention away from important professional duties, such as preparing and delivering online lessons, providing feedback on student work, and maintaining communication with colleagues, students, and parents. This reduced engagement in core responsibilities can contribute to lower job satisfaction and a sense of burnout among teachers. Therefore, the combination of pandemic-related stressors and PIU may thwart teachers’ basic psychological needs for autonomy, competence, and relatedness, as they struggle to effectively manage their time, utilize new technologies, and maintain meaningful interactions with others in their professional lives. Section title: Hypotheses for study 2: the effect of problematic internet use on psychological need thwarting of online teaching and psychological distress Educational score: 2.181476354598999 Domain: other Document type: Study Language: en According to self-determination theory (SDT), when these three basic psychological needs are not met, individuals experience frustration, leading to PNT ( 69 ). PNT encompasses the thwarting of autonomy, competence, and relatedness needs ( 69 , 70 ). For older teachers, their lack of proficiency in technology use and unfamiliarity with online teaching would increase the frustration with their own competence (i.e., competence thwarting) ( 48 , 71 ). In fact, teachers had to conduct online teaching following the rules enforced by the education sector, which may have negatively impacted teachers’ sense of autonomy (i.e., autonomy thwarting) ( 72 ). And the low teaching motivation and isolation due to online classes, suggest that they experience relatedness thwarting ( 53 ). Previous research has shown that PIU is positively correlated with PNT among primary and middle school teachers ( 44 ). Thus, we hypothesize that PIU is significantly positively correlated with PNT among older teachers. Section title: Hypotheses for study 2: the effect of problematic internet use on psychological need thwarting of online teaching and psychological distress Educational score: 1.4545371532440186 Domain: other Document type: Study Language: en Existing research has demonstrated that PNT in online teaching is significantly positively correlated with psychological distress among primary and middle school teachers ( 44 , 53 ). Moreover, Yi et al. ( 44 ) found that PIU had an indirect positive effect on psychological distress through the mediator of PNT in online teaching. Based on this, we hypothesize that among older teachers, PIU influences psychological distress through the mediator of PNT in online teaching, implying that PNT has a mediating effect between PIU and psychological distress. Section title: Hypotheses for study 3: the effect of problematic internet use on burnout and psychological distress Educational score: 2.5653438568115234 Domain: other Document type: Study Language: en As discussed in Study 1, PIU impacts the psychological distress of older teachers. Moreover, according to the Resource Model of Self-Control, PIU leads to teachers’ ego depletion and emotional exhaustion ( 66 ). Exhaustion, as a core dimension of burnout ( 73 ), has been shown to be associated with students’ PIU in numerous empirical studies ( 74 , 75 ). The positive relationship between teachers’ PIU and occupational burnout has also been demonstrated in existing research ( 52 ), with results indicating that PIU is significantly correlated with burnout among Hungarian high school teachers. Furthermore, a substantial body of literature has shown that burnout is closely related to psychological distress ( 76 , 77 ). Therefore, we postulate that in the late stage of the COVID-19 pandemic in China, PIU leads to occupational burnout among older teachers, ultimately impacting their mental health. Based on this, we further hypothesize that occupational burnout mediates the relationship between PIU and psychological distress. Section title: Participants and procedure Educational score: 2.559607744216919 Domain: other Document type: Study Language: en The pandemic outbreak resulted in the closure of educational institutions and a subsequent transition to online teaching modalities. During the critical period from August to November 2020, Study 1 was conducted utilizing convenience sampling, a method selected due to its feasibility in accessing the target population. Ethical approval for this study was obtained from the Institutional Review Board of the Jiangxi Psychological Counselors Association . The Jiangxi and Sichuan Education Bureaus assisted in data collection by inquiring about teachers’ willingness to participate. The online survey platform was designed to require completion of all questions before submission, ensuring no missing data in the final dataset. To ensure the quality and validity of the data, we implemented stringent criteria for participant inclusion. Initially, we selected participants aged between 40 and 60 years, followed by the exclusion of those with completion times of less than 120 s, which were considered inadequate to guarantee serious engagement with the survey content. After applying these criteria, from an initial pool of 9,030 participants, the refined dataset comprised 3,929 participants (29.7% male, 70.3% female; age range: 40–60 years). Supplementary Table S1 provides detailed demographic information. Section title: Measures Educational score: 2.2908647060394287 Domain: biomedical Document type: Study Language: en Study 1 used three scales to measure the variables: the Fear of COVID-19 Scale (FCV-19S), Smartphone Application-Based Addiction Scale (SABAS), and the 21-item Depression, Anxiety, and Stress Scale (DASS-21). All scale items are listed in Supplementary Table S2 . The measures are described below. Section title: Measures Educational score: 3.8757102489471436 Domain: biomedical Document type: Study Language: en The FCV-19S ( 78 ) is a 7-item measure that assesses an individual’s fear of COVID-19 using a 5-point Likert scale ranging from 1 ( “Totally disagree” ) to 5 ( “Completely agree” ). Higher scores on the FCV-19S indicate greater levels of fear. The Chinese version of the FCV-19S has been validated by Chen et al. ( 79 ) on a sample of primary and middle school teachers. The Chinese version of FCV-19S has demonstrated a unidimensional structure and strong psychometric properties ( 79 ). In the current study, the scale demonstrated good internal consistency reliability, with a Cronbach’s alpha coefficient of 0.895 and a McDonald’s omega coefficient of 0.896. Sample items include: “It makes me uncomfortable to think about Coronavirus-19.” Section title: Measures Educational score: 2.8377344608306885 Domain: biomedical Document type: Study Language: en PSU, as an indicator of PIU, was measured using the 6-item SABAS ( 80 ), which employs a 6-point Likert scale ranging from 1 ( “Strongly disagree” ) to 6 ( “Strongly agree” ). Higher scores indicate more PIU. The Chinese version of the SABAS has demonstrated a unidimensional structure and strong psychometric properties ( 81 , 82 ). In the present study, it demonstrated satisfactory internal consistency (Cronbach’s α = 0.877; McDonald’s ω = 0.880). Sample items include: “My smartphone is the most important thing in my life.” Section title: Measures Educational score: 3.9588725566864014 Domain: biomedical Document type: Study Language: en Psychological distress was assessed using the Chinese version of the DASS-21 ( 83 , 84 ). The DASS-21 uses a 4-point scale ranging from 0 ( “Never” ) to 3 ( “Almost always” ), with higher scores indicating more severe psychological distress. The scale has satisfactory psychometric properties and is considered a valid indicator of general psychological distress ( 85 , 86 ). In the current study, the internal consistency of the DASS-21 was excellent (Cronbach’s α = 0.968; McDonald’s ω = 0.968). Sample items include: “I could not seem to experience any positive feeling at all.” Section title: Data analysis Educational score: 3.9124133586883545 Domain: biomedical Document type: Study Language: en Descriptive statistics were calculated for each variable. Recommended cutoff values were used to examine the severity of fear of COVID-19 [cutoff = 17.5; ( 87 )], PSU [cutoff = 21; ( 88 )], and psychological distress [cutoff = 34; ( 89 )] among older teachers. Pearson correlation analysis was used to analyze the relationships between the variables by using SPSS 25. The Generalized Linear Model (GLM) in Jamovi 2.3.23 was employed to test the mediation effect, with psychological distress as the dependent variable, fear of COVID-19 as the independent variable, and PIU as the mediator, controlling for age and gender. Section title: Results Educational score: 3.846879720687866 Domain: biomedical Document type: Study Language: en The prevalence rates of fear of COVID-19, PIU, and psychological distress among the 3,929 older teachers in Study 1 were 28.7, 27.4, and 2.8%, respectively (see Supplementary Table S3 ). These rates represent the percentage of participants surpassing the established cutoffs for abnormal levels of fear of COVID-19, PIU, and psychological distress. All three variables were significantly and positively correlated, with coefficients ranging from 0.35 to 0.45, as detailed in Supplementary Table S3 . Notably, the strongest correlation was observed between fear of COVID-19 and psychological distress, with a coefficient of 0.45 ( p < 0.01). Section title: Results Educational score: 4.093757629394531 Domain: biomedical Document type: Study Language: en The mediation analysis showed that fear of COVID-19 was significantly associated with both PIU ( β = 0.353, 95% CI [0.486, 0.574], p < 0.001) and psychological distress ( β = 0.380, 95% CI [1.650, 1.923], p < 0.001). PIU was also significantly associated with psychological distress ( β = 0.220, 95% CI [0.598, 0.780], p < 0.001), after controlling for gender and age. The bias-corrected bootstrapping mediation test confirmed the significant indirect effect of fear of COVID-19 on psychological distress through PIU (95% CI [0.309, 0.422]). Section title: Participants and procedure Educational score: 1.8732088804244995 Domain: other Document type: Study Language: en Study 2 was conducted in November 2021 during the strict COVID-19 lockdown in China, where new cases led to local lockdown and a shift to online teaching. The online survey, approved by the Institutional Review Board of the Jiangxi Psychological Counselors Association , was voluntarily completed by teachers. The local education bureau assisted in data collection via hyperlinks to 9,242 teachers by convenience sampling. Following the inclusion of participants aged between 40 and 60 years, and then the exclusion of those with response times under 120 s, the dataset encompassed 3,502 primary and middle school teachers from a city in Jiangxi Province. The gender composition was nearly equal, with 50.1% male and 49.9% female. Supplementary Table S4 presents detailed demographic information. Section title: Measures Educational score: 1.9870012998580933 Domain: other Document type: Study Language: en As described in Study 1, the SABAS was used to measure participants’ excessive smartphone use. The internal consistency of SABAS was satisfactory in Study 2 (Cronbach’s α = 0.849; McDonald’s ω = 0.854). Section title: Measures Educational score: 1.987356185913086 Domain: other Document type: Study Language: en The Psychological Need Thwarting Scale of Online Teaching (PNTSOT) ( 44 ) was used to assess older schoolteachers’ PNT toward online teaching during the period of school closure. The PNTSOT comprises three subscales (i.e., autonomy, competence, and relatedness thwarting) and is rated on a seven-point Likert-type scale, with responses ranging from 1 ( “Strongly disagree” ) to 7 ( “Strongly agree” ) ( 70 ). Higher scores indicate more serious PNT during online teaching tasks. A sample item from the scale is “In online courses during the pandemic, I cannot decide for myself how I want to teach.” For a comprehensive list of items in the PNTSOT, refer to Supplementary Table S2 . Yi et al. ( 44 ) found sound factorial validity of the scale among schoolteachers, and the internal reliability of the PNTSOT was very good in the present study (Cronbach’s α = 0.867; McDonald’s ω = 0.869). Following Yi et al. ( 44 ), the three kinds of PNT were treated as an overall construct. Section title: Measures Educational score: 2.4060914516448975 Domain: biomedical Document type: Study Language: en The DASS-21 was used to measure psychological distress, as described in Study 1. In Study 2, Cronbach’s α = 0.865 and McDonald’s ω = 0.889. Section title: Data analysis Educational score: 3.2938284873962402 Domain: biomedical Document type: Study Language: en Following the data analysis outlined in Study 1, we conducted descriptive analyses, correlation analysis, and mediation analysis using the GLM in Jamovi 2.3.23. In Study 2, psychological distress served as the dependent variable, PIU as the independent variable, and PNT as the mediator. We controlled for age and gender as potential confounding variables. Section title: Results Educational score: 3.3886239528656006 Domain: biomedical Document type: Study Language: en In Study 2, the prevalence rates of PIU and psychological distress among the 3,502 older teachers were 27.4 and 3.9%, respectively ( Supplementary Table S5 ). The three variables (PIU, PNT, and psychological distress) were significantly and positively correlated, with coefficients ranging from 0.28 to 0.35, as presented in Supplementary Table S5 . The most robust correlation was identified between PNT and psychological distress, with a coefficient of 0.35 ( p < 0.01). Section title: Results Educational score: 3.7331383228302 Domain: biomedical Document type: Study Language: en The results of the mediation analysis showed that PIU was significantly associated with both PNT ( β = 0.276, 95% CI [0.462, 0.581], p < 0.001) and psychological distress ( β = 0.251, 95% CI [0.780, 1.001], p < 0.001) among older teachers. Additionally, PNT showed a significant positive association with psychological distress ( β = 0.281, 95% CI [0.467, 0.585], p < 0.001). A bias-corrected bootstrapping mediation test confirmed that the impact of PIU on older teachers’ psychological distress, mediated by PNT, was statistically significant (95% CI [0.230, 0.318]). Section title: Participants and procedure Educational score: 1.6277738809585571 Domain: other Document type: Study Language: en As the COVID-19 situation improved, Jiangxi Province resumed offline teaching in January 2022, and Study 3 was conducted during this period. The online survey for Study 3 was approved by the Institutional Review Board of the Jiangxi Psychological Counselors Association , and the local education bureau assisted in collecting online survey data through hyperlinks. Participation was voluntary, and a total of 4,045 primary and secondary school teachers took part in the survey. The online survey platform required all questions to be answered before submission, resulting in no missing data in the final dataset. After applying the inclusion criterion of participants aged between 40 and 60 years and then excluding those with response times less than 120 s, the final dataset comprised 1,276 individuals, with a gender distribution of 49.5% males and 50.5% females. Supplementary Table S6 presents detailed demographic information of the participants, providing insights into the composition of the older teachers group. Section title: Measures Educational score: 1.6309365034103394 Domain: other Document type: Study Language: en As in the previous studies, the SABAS was used to measure the level of PIU among older teachers. The internal consistency of SABAS in Study 3 was satisfactory (Cronbach’s α = 0.878; McDonald’s ω = 0.883). Section title: Measures Educational score: 2.31221079826355 Domain: other Document type: Study Language: en Study 3 assessed older school teachers’ occupational burnout using the “Emotional Exhaustion” subscale (eight items) of the Chinese version of the Primary and Secondary School Teachers’ Job Burnout Questionnaire (CTJBQ) ( 90 ). The CTJBQ was developed with reference to the MBI structure ( 91 ) to accommodate the cultural and linguistic background of mainland Chinese teachers. Emotional exhaustion was chosen as it contributes most significantly to burnout ( 91 , 92 ). Participants rated the frequency of their experiences on a 7-point Likert scale, ranging from 1 ( “Never” ) to 7 ( “Daily” ). An example item is “I feel that teaching is genuinely an exhausting job for me.” Detailed items of this scale are provided in Supplementary Table S2 . Higher scores indicated greater occupational burnout. Internal consistency for burnout scores was good (Cronbach’s α = 0.950; McDonald’s ω = 0.951). Section title: Measures Educational score: 2.3534040451049805 Domain: biomedical Document type: Study Language: en The DASS-21 was used to measure psychological distress, as described in Study 1 and Study 2. In Study 3, Cronbach’s α = 0.967 and McDonald’s ω = 0.967. Section title: Data analysis Educational score: 3.0585386753082275 Domain: biomedical Document type: Study Language: en Consistent with the methodologies employed in the preceding studies, Study 3 involved a series of statistical analyses, including descriptive analyses, correlation analysis, and mediation analysis using the GLM in Jamovi 2.3.23. In this analysis, we examined occupational burnout as a mediator in the relationship between PIU and psychological distress, controlling for age and gender. Section title: Results Educational score: 3.1938140392303467 Domain: other Document type: Study Language: en Study 3 found the prevalence rates of PIU and psychological distress among the 1,276 older teachers to be 24.5 and 5.6%, respectively ( Supplementary Table S7 ). PIU, occupational burnout, and psychological distress were significantly and positively correlated, with coefficients ranging from 0.43 to 0.51, as presented in Supplementary Table S7 . The strongest correlation was observed between occupational burnout and psychological distress, with a correlation coefficient of 0.51 ( p < 0.01). Section title: Results Educational score: 3.966423511505127 Domain: biomedical Document type: Study Language: en The mediation analysis showed significant positive associations between PIU and occupational burnout ( β = 0.485, 95% CI [0.846, 1.032], p < 0.001) and between PIU and psychological distress ( β = 0.245, 95% CI [0.740, 1.145], p < 0.001). PIU was also significantly associated with psychological distress ( β = 0.387, 95% CI [0.666, 0.875], p < 0.001). The bias-corrected bootstrapping mediation test confirmed the significant indirect effect of PIU on psychological distress through occupational burnout (95% CI [0.602, 0.845]). Section title: Discussion Educational score: 2.904491662979126 Domain: biomedical Document type: Study Language: en Our three-year investigation in China, comprising three distinct cross-sectional studies, examined the relationship between PIU and mental health among older teachers during the COVID-19 pandemic. Using a threshold of 21 points to identify PIU ( 89 ), we found consistently high prevalence rates across the three-year period: 27.4, 27.4, and 24.5%, respectively. These findings suggest persistent PIU concerns among older teachers, though it’s important to note these rates come from different participant cohorts. Section title: Discussion Educational score: 3.713228225708008 Domain: biomedical Document type: Study Language: en Comparing our findings with other studies on PIU during the pandemic reveals both similarities and differences. Our results align with Zhao et al. ( 16 ), who reported a PIU prevalence of 28.4% among Chinese university students. Mohler-Kuo et al. ( 17 ) found a slightly lower prevalence of 21.3% among Swiss young adults. However, two Hungarian studies ( 35 , 36 ) reported significantly lower PIU prevalence rates, below 6%. These variations highlight the importance of context-specific analyses in understanding PIU patterns during the pandemic. Section title: Discussion Educational score: 2.2484183311462402 Domain: biomedical Document type: Study Language: en The cultural context of our study may also play a role in the prevalence of PIU. In Asian countries, cultural norms might limit opportunities for free interaction and self-expression ( 93 ), leading to increased reliance on mobile devices. This cultural backdrop could explain the high PIU prevalence observed in our study, as individuals may have used the internet to cope with pandemic-induced stress and isolation, particularly among older adults. This insight underscores the importance of considering cultural factors when examining internet usage behaviors during crises. Section title: Discussion Educational score: 1.5195865631103516 Domain: other Document type: Other Language: en The compensatory internet use theory ( 6 ) offers a valuable perspective for understanding the connection between fear of COVID-19 and PIU. It suggests that individuals may turn to the internet as a way to fulfill psychological needs that are not being met in their offline lives. Consequently, the stress and isolation exacerbated by the pandemic could potentially drive individuals to seek solace in internet engagement as a means to mitigate their fears regarding COVID-19. Section title: Discussion Educational score: 3.9136505126953125 Domain: biomedical Document type: Study Language: en Furthermore, our study consistently demonstrated the significant relationship between PIU and psychological distress among older teachers, even after controlling for gender and age. These results align with previous research that has established the negative impact of PIU on mental health ( 22–27 ), including anxiety, depression and suicidal thoughts. The Resource Model of Self-Control ( 66 ) provides a theoretical framework to understand this relationship, suggesting that PIU depletes individuals’ limited self-control resources, leading to ego depletion and ultimately resulting in psychological distress ( 73 ). Section title: Discussion Educational score: 2.666722536087036 Domain: other Document type: Study Language: en Building upon the direct effect of PIU on psychological distress, this study also revealed the mediating roles of PNT and occupational burnout in the relationship between PIU and psychological distress. These findings are consistent with self-determination theory ( 69 ) and previous research on the relationship between PIU and burnout ( 53 ) and the association between burnout and psychological distress ( 76 , 77 ). The thwarting of basic psychological needs for autonomy, competence, and relatedness in online teaching, as well as the experience of occupational burnout during the transition back to offline teaching, serve as important mechanisms through which PIU contributes to psychological distress among older teachers. Section title: Discussion Educational score: 2.3289594650268555 Domain: other Document type: Study Language: en Notably, despite different participants, the impact coefficient of PIU on the psychological distress of older teachers generally showed a stable trend, with β values ranging from 0.220 (Study 1), 0.251 (Study 2), to 0.245 (Study 3). This demonstrated that the harm caused by PIU to the mental health of this population remained consistent, suggesting that for older teachers, the adverse impact of PIU on psychological distress is persistent, and should draw the attention of relevant educational departments. Section title: Discussion Educational score: 2.793632745742798 Domain: other Document type: Study Language: en Our findings suggest a complex interplay of factors associated with PNT and occupational burnout among educators during different phases of the pandemic. Beyond our primary variables of interest, research indicates several other important factors in understanding teacher wellbeing. Bartholomew et al.’s study ( 94 ) showed associations between job pressure and the thwarting of autonomy, competence, and relatedness needs among physical education teachers. Chen et al. ( 95 ) found correlations between teachers’ perceived instructional leadership and burnout levels. School environment has also been linked to teacher burnout patterns ( 96 ). These findings indicate that the relationships between PIU, PNT, and burnout exist within a broader context of organizational and environmental factors. Understanding these multifaceted relationships could be particularly relevant when examining teachers’ experiences across different pandemic phases—from initial adjustment to online teaching through the return to in-person instruction. Future research would benefit from examining this broader range of variables to provide a more comprehensive understanding of teachers’ psychological wellbeing during periods of educational transition. Section title: Implications Educational score: 1.8551254272460938 Domain: other Document type: Other Language: en The findings of this study have important implications for the wellbeing of older teachers in China and beyond. Given the significant impact of PIU on psychological distress, it is crucial to develop and implement targeted interventions to promote healthy smartphone use and mitigate the negative effects of PSU on mental health. These interventions should not only focus on reducing excessive smartphone use but also address the underlying psychological factors that contribute to psychological distress, such as the thwarting of basic psychological needs and the experience of occupational burnout. Additionally, interventions should be sensitive to the cultural context in which they are implemented, taking into account the unique challenges and pressures faced by teachers in different settings. Section title: Implications Educational score: 1.3555158376693726 Domain: other Document type: Other Language: en In light of these considerations, we propose the following strategic directions. Firstly, educational institutions and policymakers should consider integrating digital literacy and mental health education into professional development programs for teachers. This would equip them with the skills to navigate the digital landscape responsibly and maintain a healthy balance between professional and personal smartphone use. Secondly, there is a need for the establishment of support systems within schools and educational communities, such as counseling services and peer support groups, to address the psychological distress experienced by teachers. These systems should be designed to identify and mitigate the factors contributing to occupational burnout and the thwarting of psychological needs. Lastly, policymakers should advocate for research-informed policies that promote a healthy work-life balance for educators, including guidelines on reasonable working hours and the responsible use of digital technologies in educational settings. Section title: Strength and limitation Educational score: 3.3493921756744385 Domain: other Document type: Study Language: en This study offers several notable methodological contributions. The repeated cross-sectional design captured data across three distinct phases of the COVID-19 pandemic, providing snapshots of PIU and psychological wellbeing among older teachers over time. The geographically diverse sample from various regions in China allowed for examination of PIU patterns across different educational contexts. Additionally, the use of validated measurement instruments with demonstrated reliability, combined with substantial sample sizes at each time point, strengthens confidence in the observed relationships between PIU, psychological need thwarting, occupational burnout, and psychological distress. Section title: Strength and limitation Educational score: 4.00682258605957 Domain: biomedical Document type: Study Language: en Despite these strengths, the study has several limitations that should be acknowledged. First, despite employing a repeated cross-sectional survey methodology, the absence of a longitudinal design constrains our capacity to delve into the developmental trajectories of the PIU prevalence and its psychological distress implications. Additionally, the cross-sectional nature of our research precludes the drawing of causal inferences, thus necessitating a cautious consideration of the potential for reverse causality. To address these limitations and to elucidate the causal pathways, future research should adopt a longitudinal design. This approach would facilitate a more profound understanding of the relationship between PIU and mental health of older teachers, allowing for a clearer delineation of the direction and strength of the relationships under investigation. Second, while the sample includes teachers from various regions in China, it may not be fully representative of the entire population of older teachers nationwide. The convenience sampling approach, facilitated by the cooperation of local Education Bureaus, was utilized to recruit participants, which could potentially limit the generalizability of our findings. Future studies should recruit larger and more diverse samples from a wider range of regions to enhance the generalizability of the findings and further explore potential regional differences. Third, the study relies on self-report measures, which may be subject to response biases such as social desirability or recall bias. Participants may have underreported their levels of PSU or psychological distress due to social stigma or a lack of awareness of their own behaviors and experiences. | Study | other | en | 0.999996 |
PMC11695433 | Section title: Introduction Educational score: 3.9786465167999268 Domain: biomedical Document type: Review Language: en Cardiovascular disease (CVD) remains a significant public health concern and is the primary cause of morbidity and mortality among the major non-communicable diseases, despite significant progress in prevention and management of CVD being made over the past half-century. Data from the AHA Heart Disease and Stroke Statistical Update indicate that approximately 28.6 million American adults were affected by some form of CVD between the late 2010s and 2020 ( 1 ). Concurrently, CVD accounted for approximately 18.6 million deaths globally in 2019, representing nearly one-third of global mortality, with approximately 870,000 of those deaths occurring in the U.S ( 2 ). This number is predicted to increase to more than 23.6 million people who will die annually by 2030 ( 3 ). Consequently, there is an urgent need to identify CVD patients at high mortality risk and to develop clinical interventions aimed at preventing adverse outcomes. Section title: Introduction Educational score: 4.1759934425354 Domain: biomedical Document type: Review Language: en Insulin resistance (IR) serves as a component of cardiovascular metabolic abnormalities, which are commonly referred to as “IR syndrome” or “metabolic syndrome (MetS).” IR accelerates the development of atherosclerosis and hypertension ( 4 ) and is closely associated with diabetes ( 5 ), stroke ( 6 ), and coronary heart disease ( 7 ). A growing body of evidence suggests that IR is closely linked to increased mortality risk, including both all-cause and cardiovascular mortality. However, the relationship between IR and mortality risk is not uniform across all population groups, and significant differences have been observed based on age, gender, and ethnicity. The association between IR and mortality risk appears to be particularly pronounced in elderly populations ( 8 ), postmenopausal women ( 9 ), and African Americans and Hispanics ( 10 ). These findings highlight the need to consider population-specific differences when evaluating the risk posed by insulin resistance. Understanding how IR contributes to mortality risk in various demographic groups may help refine risk assessment strategies and develop targeted interventions for reducing the burden of CVD and premature mortality. Section title: Introduction Educational score: 4.817343235015869 Domain: biomedical Document type: Review Language: en The hyperinsulinemic-euglycemic clamp test (HEC) and the homeostasis model assessment of insulin resistance (HOMA-IR) are common techniques for determining IR ( 11 ). However, due to the complexity and cost of the process, as well as the fact that insulin levels are not routinely measured in clinical practice, both approaches are ineffective for large-scale epidemiological studies. Hence, some non-insulin-based IR metrics, such as the triglyceride to high-density lipoprotein cholesterol (TG/HDL-c) ratio and the triglyceride-glucose index (TyG index), have been developed. However, existing indices do not account for the effects of nutritional intake and metabolic status, such as body mass index (BMI), thereby limiting the development of effective clinical disease prediction models. METS-IR, a novel index for assessing insulin resistance (IR), strongly correlates with HEC ( 12 ). It combines fasting blood glucose (FBG), triglycerides (TG), high-density lipoprotein (HDL-c) and BMI, offering a cost-effective and accessible alternative for large-scale screening and ongoing patient monitoring. Previous studies have demonstrated that METS-IR is closely associated with hypertension ( 13 ) and hyperuricemia ( 14 ). Additionally, METS-IR is more effective at identifying people developing MetS and T2DM at an early stage than other insulin-dependent indices ( 15 ). Emerging evidence suggests that METS-IR is also linked to important cardiovascular indicators, such as coronary artery calcification and subclinical myocardial injury ( 16 ), which are early markers of cardiovascular damage. This association highlights the broader impact of METS-IR on cardiovascular health and its potential to predict adverse cardiovascular outcomes. In rural China, baseline METS-IR levels and their long-term trends have been strongly associated with increased risks of CHD and stroke ( 17 ). Furthermore, METS-IR has demonstrated superior accuracy in predicting diabetes at various future time intervals in the Japanese population, compared to the TyG index, TyG-BMI, and TG/HDL-c ratio ( 18 ). METS-IR has also shown a more significant association with all-cause and cardiovascular mortality in the U.S. population compared to the TyG index, TG/HDL-c, and HOMA-IR ( 19 ). As an innovative tool for assessing IR, METS-IR utilizes multiple clinical parameters without the need for fasting insulin, making it more accessible for primary care, community settings, and large-scale studies. By incorporating BMI and HDL-c, METS-IR provides a comprehensive view of metabolic health and facilitates early cardiovascular risk detection, suggesting that it could serve as a superior risk stratification tool in clinical practice. However, its association with mortality in individuals with CVD remains unexplored. We hypothesize that METS-IR will be a significant predictor of mortality in individuals with CVD. This hypothesis is based on the ability of METS-IR to integrate critical metabolic parameters, potentially offering useful prognostic value in CVD individuals. Hence, we conducted this study to investigate the prognostic value of METS-IR for mortality risk in CVD patients. Section title: Study design and participants Educational score: 2.8799526691436768 Domain: biomedical Document type: Study Language: en The National Health and Nutrition Examination Survey (NHANES) is a population-based, cross-sectional survey designed to collect information on the health and nutritional status of the U.S. household population. Using a multistage probability sampling methodology, the data were collected through laboratory tests, in-home structured interviews, and mobile center physical examinations. This research was conducted in accordance with the guidelines outlined in the Declaration of Helsinki. Ethics approval for the study was obtained from the Ethics Committee of the National Center for Health Statistics. Written informed consent was provided by all participants prior to their involvement in this study. Section title: Study design and participants Educational score: 3.7394917011260986 Domain: biomedical Document type: Study Language: en We collected data from participants interviewed between 1999 and 2018. Subsequently, a longitudinal follow-up cohort was created within the NHANES dataset as a result of using the National Death Index (NDI) from the National Center for Health Statistics (NCHS) to ascertain the participants’ survival outcomes. Individuals was diagnosed with CVD if they indicated in a validated questionnaire that they had ever been informed by a physician or other health care provider that they had congestive heart failure, coronary heart disease, angina pectoris, or stroke. More detailed information about self-report questionnaires can be viewed on this website at https://www.cdc.gov/nchs/data . Section title: Study design and participants Educational score: 2.520125389099121 Domain: biomedical Document type: Study Language: en After removing respondents who did not provide baseline information on FBG, TG, HDL-c, or BMI or who did not provide follow-up information, a cohort of 2515 patients diagnosed with CVD was chosen for the study. Figure 1 depicts the complete data selection procedure. After confirming that each participant gave their informed consent, the NCHS Ethics Committee approved the survey. Section title: Measurement of METS-IR Educational score: 4.036413669586182 Domain: biomedical Document type: Study Language: en The METS-IR was computed using the following formula ( 12 ): ln(2 × FBG [mg/dL] + TG [mg/dL]) × BMI (kg/m^2)/ln(HDL-c [mg/dL]). Blood parameters were obtained from venous blood samples collected from participants after an overnight fast of at least 8 hours. FBG and TG levels were enzymatically analyzed using a Roche Modular P chemistry analyzer. BMI was calculated using a formula based on height and weight. Section title: Study outcomes Educational score: 3.933542013168335 Domain: biomedical Document type: Study Language: en The ICD10 code was used to define the outcome variables, which included all-cause and cardiovascular mortality. Follow-up time was calculated from the date of the NHANES examination to the date of death or December 31, 2019, whichever occurred first. All-cause mortality was determined using the records from NDI, which were connected to the NHANES datasets. Deaths attributed to heart diseases (ICD-10 codes I00-I09, I11, I13, I20-I51) or cerebrovascular diseases (ICD-10 codes I60-I69) were categorized as cardiovascular mortality ( 20 ). More detailed information about mortality factors can be viewed on this website at https://www.cdc.gov/nchs/data-linkage/mortality.htm . Section title: Covariates Educational score: 4.122664451599121 Domain: biomedical Document type: Study Language: en The participants’ demographic information, medical history, and results from laboratory blood tests were collected. Age, gender, race, education level, marital status, and income-to-poverty ratio (PIR) were among the demographic data. Race/ethnicity was classified into five groups: non-Hispanic White, non-Hispanic Black, Mexican American, other Hispanic, and other races. The three categories of education levels were below high school, high school or equivalent, and college or above. Marital status was categorized as living with a partner or not. Every participant completed a thorough questionnaire about their medical history and lifestyle choices. Based on their smoking status, the participants were divided into three groups: never smoked, former smokers, and current smokers. Individuals who consumed at least 12 drinks within the past 12 months were identified as alcohol users. A questionnaire about participation in vigorous or moderate work or recreational activities was used to measure physical activity (PA). Medical history information included hypertension, diabetes, CVD, and cancer. The criteria for hypertension included a self-reported history of hypertension, oral antihypertensive medication use, or a systolic blood pressure of at least 140 mmHg or a diastolic blood pressure of at least 90 mmHg. The American Diabetes Association (ADA) diagnostic criteria for diabetes state that diabetes can be identified by glycated hemoglobin A1c (HbA1c) ≥ 6.5 percent, fasting glucose ≥ 7.0 mmol/L, use of insulin or oral hypoglycemic medication, or self-reported diagnosis ( 21 ). Heart attack, stroke, angina, coronary heart disease, and self-reported heart failure were all included in the CVD history. Laboratory blood test data included fasting glucose, fasting insulin, high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), total cholesterol (TC), TG, HbA1c, serum creatinine, eGFR (estimate glomerular filtration rate), and serum uric acid. Measurements were made to determine BMI, SBP, and DBP. The eGFR calculation in this study was based on the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation ( 22 ). Section title: Statistical analysis Educational score: 4.130232810974121 Domain: biomedical Document type: Study Language: en We used the fasting subsample weights and made adjustments to account for multiple cycles. Frequencies and percentages are used to represent categorical variables, and weighted means with standard error are used to represent continuous variables. For the two cycles from 1999 to 2002, the fasting subsample Mobile Examination Center (MEC) weight for 4-year was multiplied by 1/5 to determine the sample weights; for the eight cycles from 2003 to 2018, the fasting subsample 16-year MEC weight was multiplied by 4/5. The METS-IR values underwent logarithmic transformation (ln METS-IR) to account for left skewness. Based on ln METS-IR quartiles (Q1–Q4), four groups of study participants were identified: quartile 1 (<3.624), quartile 2 (3.624-3.791), quartile 3 (3.791-3.978), and quartile 4 (>3.978). A one-way ANOVA was used for continuous variables and a Pearson chi-square test was used for categorical variables in order to compare baseline characteristics between quartile groups. A multivariate Cox regression model was employed to further investigate the impact of METS-IR levels on the risk of all-cause and cardiovascular mortality in CVD patients, with outcomes reported as hazard ratios (HRs) and 95% confidence intervals (CIs). The selection of potential covariates for the multivariable regression models was based on the following criteria: (1) relevant demographic characteristics; (2) variables shown to affect METS-IR and CVD in previous studies ( 19 ); (3) variables whose inclusion resulted in a change of more than 10% in the coefficients of the basic model, in accordance with the STROBE statement ( 23 ), the basic model changes by more than 10% after the introduction of covariates; and (4) other variables accumulated from clinical experience, including factors that could influence the outcomes but were not captured in the previous categories. To verify that the proportional hazards assumption was satisfied, we conducted the Schoenfeld residual test . This test assesses whether the hazard ratios are proportional over time for each covariate in the model. In Model 1, there was no adjustment; in Model 2, age, gender, and race were adjusted for; in Model 3, variables such as age, gender, race, education level, smoking status, married status, alcohol drinking, BMI, waist circumference, PIR, PA, LDL-c, TC, HbA1c, eGFR, hypertension, and diabetes status were included. To assess the nonlinear relationship between METS-IR and mortality, multivariable restricted cubic splines (RCS) were employed. We chose 3 assumed knots based on the recommendations of Harrell ( 24 ), the complexity of the relationship between METS-IR and mortality, Akaike Information Criterion (AIC) ( 25 ), and prior similar studies ( 26 ). Additionally, the relationship between METS-IR and mortality was examined using a two-piecewise Cox proportional hazards model on either side of the inflection point. Stratified analyses were also performed by age (< 60 or ≥ 60 years), gender (male or female), BMI (< 25, 25-30, or ≥ 30 kg/m 2 ), hypertension (yes or no), diabetes (yes or no), physical activity (yes or no), and smoker (current, former, or never). Missing covariate data were imputed via the application of multiple imputation (MI) using chained equation (MICE) methodology. Details of the proportions of missing covariates and the methodology of MI are presented in Supplementary Table S1 . Two sensitivity analyses were conducted to assess model stability. To minimize the impact of missing data on the main results, participants with missing values were removed from consideration. In addition, participants who were diagnosed with cancer at the start of the study were excluded to reduce potential confounding effects. All statistical analyses were performed using R software (version 4.3.1) and EmpowerStats software. Statistical significance was defined as a two-tailed P-value < 0.05. Section title: Baseline characteristics Educational score: 4.105485916137695 Domain: biomedical Document type: Study Language: en Table 1 shows the baseline characteristics of participants stratified by lnMETS-IR quartile. The average age of participants was 66.65 years, with 56.10% were male. The mean METS-IR was 46.36 ± 12.82. During a median follow-up of 91.44 months, the rates of all-cause mortality and cardiovascular mortality were 43.34% and 17.77%, respectively. As shown in Table 1 , compared to patients in the lowest quartile, those with higher lnMETS-IR were typically younger, predominantly male, and had a higher prevalence of hypertension, diabetes, and heart failure. They also demonstrated lower levels of HDL-c, but higher waist circumference, BMI, fasting glucose, fasting insulin, triglycerides, HbA1c, and serum uric acid (all P < 0.05). Section title: Association of METS-IR with mortality Educational score: 4.143831729888916 Domain: biomedical Document type: Study Language: en The proportional hazards assumption was assessed using the Schoenfeld residuals test. The test results indicated that the assumption holds for all covariates included in the model (p > 0.05 for all variables), suggesting that the hazard ratios for these variables remain constant over time. Table 2 shows the number of deaths throughout the follow-up period: 1090 from all causes, and 447 from cardiovascular-related causes. Variables selected for inclusion in the multivariable regression models were based on their relevance to the study outcomes, changes in model coefficients, and clinical significance. Specifically, covariates that resulted in a greater than 10% change in the coefficients of the basic model, as well as those supported by prior literature, were included in the Model 3. According to the results of a multivariate Cox proportional hazard regression analysis, in continuous models, after adjusting for all covariates in Model 3, there was no significant association between lnMETS-IR and the risk of all-cause mortality and cardiovascular mortality, with HRs of 0.97 (0.63, 1.50) and 1.13 (0.57, 2.24). In categorical models, the HRs and 95% CIs for all-cause mortality were 1.00 (reference) for Q1, 0.76 (0.64, 0.91) for Q2, 0.82 (0.67, 1.00) for Q3, and 0.98 (0.75, 1.27) for Q4, with no significant trend (P for trend = 0.715). For cardiovascular mortality, the HRs and 95% CIs were 1.00 (reference) for Q1, 0.75 (0.57, 0.99) for Q2, 0.87 (0.63, 1.19) for Q3, and 1.13 (0.76, 1.69) for Q4, with no significant trend (P for trend = 0.662). Section title: Association of METS-IR with mortality Educational score: 4.157398223876953 Domain: biomedical Document type: Study Language: en Cox proportional hazards regression models with restricted cubic splines (RCS) and smooth curve fitting using the penalized spline approach were employed. It is interesting to note that U-shaped relationships were found between lnMETS-IR and all-cause mortality and cardiovascular mortality . Segmented Cox regression analysis revealed inflection points for lnMETS-IR in correlation with all-cause and cardiovascular mortality at 3.70 and 3.67, respectively, suggesting that METS-IR exerts a bidirectional effect within specific ranges ( Table 3 ). In the lower range of the inflection points, METS-IR was found to be in negative correlation with both outcomes. The risk of all-cause mortality was observed to decline by 75% with each one-unit increase in lnMETS-IR (HR: 0.25, 95% CI: 0.14–0.46, P < 0.001), and a 79% reduction in the risk of cardiovascular mortality was found with each one-unit increase in lnMETS-IR (HR: 0.21, 95% CI: 0.07–0.56, P < 0.001). Conversely, above the inflection points, METS-IR exhibited a positive significant correlation with both outcomes. Each one-unit increase in lnMETS-IR was correlated with a 1.80 elevation in all-cause mortality (HR: 2.80, 95% CI: 1.61–4.88, P < 0.001) and a 2.33 elevation in cardiovascular mortality (HR: 3.33, 95% CI: 1.43–7.75, P = 0.005). Section title: Association of METS-IR with mortality Educational score: 4.078049659729004 Domain: biomedical Document type: Study Language: en In addition, we explored the correlation between METS-IR and mortality stratified by heart failure, coronary heart disease, heart attack, angina, and stroke, as showed in Figures 3 , 4 . METS-IR exhibited an L-shaped or U-shaped association with all-cause mortality among these five groups . Similarly, an L-shaped or U-shaped association with all-cause mortality was also found among heart failure, coronary heart disease, heart attack, and angina patients, while no significant nonlinear association was observed among stroke patients (P for nonlinear = 0.244) . Section title: Stratified analyses Educational score: 4.076540946960449 Domain: biomedical Document type: Study Language: en The survival advantage correlated with a higher lnMETS-IR (≥ 3.70 for all-cause mortality and ≥ 3.67 for cardiovascular mortality) compared to a lower lnMETS-IR (< 3.70 for all-cause mortality and < 3.67 for cardiovascular mortality) in CVD individuals remained consistent across various subgroups stratified by age, gender, BMI, PA, diabetes, smoking status, and hypertension (all P for interaction > 0.05), as shown in Figure 5 . No significant interaction was identified between METS-IR and any of the stratified variables. Section title: Sensitivity analysis Educational score: 4.021451473236084 Domain: biomedical Document type: Study Language: en In the sensitivity analyses, after excluding individuals with incomplete baseline covariate data, a total of 2129 participants were included. The results indicated that the relationships between METS-IR and both all-cause and cardiovascular mortality were stable (see Supplementary Table S2 ). Furthermore, after the exclusion of patients who declared a cancer diagnosis at the outset of the study, the remaining cohort contained 2112 individuals. After adjustment for all confounders, the associations between METS-IR and mortality remained consistent (see Supplementary Table S3 ). Section title: Discussion Educational score: 4.0640153884887695 Domain: biomedical Document type: Study Language: en To the best of our knowledge, this is the first study to reveal a U-shaped relationship between lnMETS-IR and the risks of all-cause and cardiovascular mortality in patients with CVD, with thresholds identified at 3.70 and 3.67. Interaction and sensitivity analyses indicated that these findings are robust. These findings suggest that METS-IR may be a useful predictor of mortality in patients with CVD. Further studies are needed to validate its clinical applicability. Section title: Discussion Educational score: 4.284994125366211 Domain: biomedical Document type: Study Language: en METS-IR is an emerging biomarker with numerous advantages in clinical practice. Unlike the HEC method, the calculation of METS-IR requires only the measurement of fasting blood glucose, lipid profiles, height, and weight. Therefore, METS-IR serves as an easily accessible, cost-effective, and practical method for evaluating IR in patients with CVD. Since its introduction, several studies have highlighted the potential of METS-IR as a robust predictor of metabolic and cardiovascular outcomes. For instance, in Japan, METS-IR was found to independently predict future type 2 diabetes in non-obese adults ( 27 ). In Korea, METS-IR showed a stronger predictive value for ischemic heart disease (IHD) in non-diabetic individuals compared to metabolic syndrome (MetS) ( 16 ). Moreover, METS-IR was positively associated with the risk of cardiovascular events in hypertensive patients ( 28 , 29 ) and demonstrated a “J-shaped” relationship with stroke and ischemic stroke risk in individuals with hypertension and obstructive sleep apnea ( 30 ). Notably, a study by Zhou et al. in heart failure patients showed that higher METS-IR levels were associated with increased mortality risk, with adjusted hazard ratios for all-cause and cardiovascular deaths being 2.48 (95% CI: 2.10–2.93) and 2.29 (95% CI: 1.83–2.87), respectively ( 26 ). METS-IR exhibits a nonlinear “U-shaped” relationship with mortality in the general population, with stronger associations than other insulin resistance markers like TyG index, TG/HDL-c, and HOMA-IR ( 19 ). These findings suggest that METS-IR may have unique prognostic value across different populations, particularly in those with hypertension, heart failure, or diabetes, underscoring the need for further studies to explore its utility in diverse patient groups. Nevertheless, no research has examined the relationship between METS-IR and the mortality risk in CVD patients. Comparison with previous studies on METS-IR and cardiovascular outcomes, our study reaches conclusions consistent with prior findings. Additionally, we extend this work by emphasizing the prognostic value of METS-IR in a broader CVD population. This study provides the first demonstration of a U-shaped relationship between lnMETS-IR and both all-cause and cardiovascular mortality. Our analysis confirmed that the proportional hazards assumption was met for all covariates (p > 0.05), supporting the validity of the Cox proportional hazards model for our data. We also analyze specific CVD subtypes, including heart failure, coronary heart disease, heart attack, and angina. Furthermore, through subgroup analyses based on different BMI categories, we highlight the broader utility of METS-IR in predicting metabolic and cardiovascular risks. Section title: Discussion Educational score: 4.1005659103393555 Domain: biomedical Document type: Study Language: en In agreement with previous studies ( 31 , 32 ), the results of this study demonstrated a U-shaped relationship between METS-IR and mortality in patients with CVD. Notably, the inflection point for all-cause mortality was approximately 3.70, while the inflection point for cardiovascular mortality was approximately 3.67. This identification of threshold values enables physicians to focus on specific patient subgroups for closer monitoring and timely intervention. In addition, we further explored the relationship of METS-IR in specific CVD subgroups and confirmed that the effect of METS-IR on mortality was consistent across CVD subgroups, showing either a J- or U-shaped relationship, except for cardiovascular mortality in the stroke population, which did not show a significant nonlinear relationship. We consider that the inconsistency of this result may be due to the small sample size of the included stroke population. Section title: Discussion Educational score: 4.180673122406006 Domain: biomedical Document type: Study Language: en An increasing number of studies indicate that metabolic factors may be associated with a worse prognosis in patients with CVD. The impact of BMI on the prognosis of patients with CVD remains a topic of active debate in the scientific community. Studies have demonstrated a J- or U-shaped correlation between BMI and the risk of developing cardiovascular events or all-cause mortality ( 33 , 34 ). Similarly, low BMI is found to be associated with elevated levels of BNP and N-terminal pro-B-type natriuretic peptide in patients with chronic heart failure, which may result in a poorer prognosis, particularly when chronic heart failure progresses to cardiac cachexia, which can further impact survival outcomes ( 35 ). In the subgroup analysis, we observed that when the lnMETS-IR value exceeded a threshold, its impact on mortality appeared to be more pronounced in individuals with a BMI < 25. We hypothesize that obese patients may manifest symptoms earlier, leading to timely intervention and optimal pharmacotherapy, while benefiting from the metaboloprotective effects of adiposity and enhanced metabolic reserves. Conversely, weight loss in individuals at elevated cardiovascular risk may be attributed to cachexia associated with severe comorbidities, such as malignancies or chronic infections ( 29 ). Section title: Discussion Educational score: 4.186990737915039 Domain: biomedical Document type: Study Language: en This finding likely reflects the complex interplay between metabolic function and mortality risk. When METS-IR is low, despite mild IR, the mortality risk remains elevated, particularly in low-BMI individuals, who typically have lower metabolic reserves and are more vulnerable to acute illnesses or chronic metabolic disturbances ( 36 ). In contrast, high METS-IR values are generally associated with increased IR, particularly in obese individuals. The accumulation of visceral fat not only exacerbates IR ( 37 ) but also promotes chronic low-grade inflammation through the secretion of pro-inflammatory cytokines such as TNF-α and IL-6 ( 38 ), which further increases the risk of cardiovascular events and endothelial dysfunction, ultimately elevating mortality risk. Section title: Discussion Educational score: 4.411505699157715 Domain: biomedical Document type: Study Language: en It is noteworthy that there is a correlation between lower IR levels and lower fasting blood glucose levels ( 39 , 40 ). Studies have demonstrated that episodes of hypoglycemia and rapid fluctuations in blood glucose levels lead to an increase in the levels of counter-regulatory hormones, including epinephrine and norepinephrine. These hormones induce platelet aggregation and vasoconstriction, which may accelerate ischemia in the cardiovascular system ( 41 ). Extremely low triglyceride and fasting glucose levels could indicate inadequate nutritional status. A J-shaped relationship has been identified between blood glucose levels and cardiovascular events or all-cause mortality. Specifically, it has been revealed that the lower the fasting blood glucose level, the worse the prognosis ( 42 ). A study conducted in Korea demonstrated that severe hypoglycemia was associated with an elevated risk of cardiovascular events and all-cause mortality in patients with T2DM ( 43 ). Similarly, decreased TG levels were found to be associated with a higher risk of developing hemorrhagic stroke among women, and had been found to serve as a predictor of cardiac death in heart failure patients ( 44 ). We discussed the potential mechanisms behind this U-shaped relationship. Significantly, the METS-IR provides valuable information on lipid metabolism abnormalities, as well as serves as an indicator of oxidative stress levels and cardiovascular risk that surpasses conventional lipid assessments. This enhanced capability may lead to a more comprehensive understanding of cardiometabolic risk factors associated with CVD outcomes. Our findings are consistent with previous studies, which also observed a U-shaped ( 19 ) or J-shaped ( 31 ) relationship between METS-IR and mortality. We believe this U-shaped relationship has important clinical implications and suggests that when evaluating metabolic health, it is crucial to consider the full spectrum of metabolic states, rather than focusing solely on absolute METS-IR values. Thus, METS-IR could be a useful tool for assessing IR and predicting outcomes in individuals with CVD. Section title: Discussion Educational score: 4.03715705871582 Domain: biomedical Document type: Study Language: en In addition to the direct link between METS-IR and mortality, clinical phenotypes such as coronary artery calcification ( 45 ), subclinical myocardial injury ( 46 ), and other cardiovascular risks ( 47 ) may mediate this relationship. METS-IR is associated with increased arterial stiffness, subclinical atherosclerosis, and endothelial dysfunction ( 48 ), all of which are key factors in CVD progression and mortality risk. These clinical manifestations highlight the importance of considering not only metabolic markers but also cardiovascular pathophysiology when evaluating mortality risk in CVD patients. Future studies should investigate the role of these phenotypes in mediating the METS-IR mortality association. Section title: Discussion Educational score: 4.784867286682129 Domain: biomedical Document type: Study Language: en The exact mechanism by which METS-IR leads to increased mortality remains unclear. IR contributes to CVD mainly by disrupting glucose and lipid metabolism, increasing vascular stiffness and endothelial dysfunction, and inducing oxidative stress and inflammatory responses. Firstly, the migration of smooth muscle cells and deposition of collagen at sites of damaged endothelial cells are thought to be mediated by elevated levels of oxidative stress associated with IR. Secondly, insulin-mediated nitric oxide (NO) production is conducive to circulation and glucose utilization. A reduction in nitric oxide production due to IR in the endothelium serves to further impair endothelial function and increase damage to endothelial cells ( 48 , 49 ). Thirdly, insulin resistance reduces insulin sensitivity in the liver and muscle tissue, thereby impeding insulin utilization and glucose uptake ( 50 ). This condition leads to hyperglycemia, exacerbation of the local inflammatory response, smooth muscle cell proliferation, collagen deposition and ultimately vascular ageing and hardening ( 51 ). Fourthly, IR has the potential to trigger thrombosis and activate platelets through mechanisms such as upregulation of the expression of adhesion-inducing factor and thromboxane A2-dependent tissue factor and activation of fibrinogen activator inhibitor-1 ( 52 , 53 ). In combination, these physiological processes contribute to the onset and progression of CVD, ultimately resulting in poor clinical outcomes. Section title: Discussion Educational score: 4.164883613586426 Domain: biomedical Document type: Study Language: en METS-IR has significant potential for clinical risk stratification and personalized interventions, especially for patients at high risk of metabolic diseases, cardiovascular conditions, and type 2 diabetes. By identifying individuals at greater metabolic risk, it can guide clinicians in tailoring preventive measures and therapies, such as lifestyle modifications and early pharmacological treatments. METS-IR could also be integrated into routine clinical screenings to identify high-risk individuals early, allowing for timely intervention. Future research should focus on validating METS-IR across diverse populations and exploring its integration into digital health tools to improve accessibility and clinical utility. However, there are several limitations that should be considered. First, as a cross-sectional study, we do not establish a causal relationship between METS-IR and mortality, and larger-scale studies are needed to validate these results. Second, the potential impact of fluctuations in METS-IR over time on its correlation with mortality remains unclear due to the lack of continuous monitoring of the METS-IR in our study. Third, residual confounders, such as long-term lifestyle habits, dietary patterns, genetic susceptibility, and environmental factors, may still influence the results. For example, individuals’ eating habits, physical activity levels, and other health behaviors may change over time, significantly impacting their metabolic status and contributing to variations in METS-IR. Future research could expand on these potential confounders by incorporating genetic data, environmental exposures, and more comprehensive behavioral assessments. Additionally, longitudinal study designs could more closely track participants’ lifestyle factors and analyze their cumulative effects over time. Moreover, the presence of CVD was self-reported and certain lifestyle variables were based on questionnaires, which introduced the possibility of recall bias or inaccuracies in the data. Future studies should incorporate more objective measures to reduce this bias. Section title: Conclusions Educational score: 4.059443950653076 Domain: biomedical Document type: Study Language: en Our findings show that METS-IR is a valuable tool for predicting all-cause and cardiovascular mortality in CVD patients. Particularly noteworthy is the presence of a U-shaped relationship between the METS-IR and both all-cause and CVD mortality. Furthermore, the identified threshold could serve as a target for interventions aimed at reducing the risk of premature mortality. Further research is required to investigate whether interventions targeting METS-IR can result in better clinical outcomes for individuals in the future. | Review | biomedical | en | 0.999996 |
PMC11695462 | Section title: Introduction Educational score: 3.9146835803985596 Domain: biomedical Document type: Review Language: en Pneumoconiosis is a parenchymal lung disease caused by inhaling various mineral dust, causing parenchymal lung reactions, resulting in fibrotic or nonfibrotic parenchymal lesions . Despite a decline in its incidence rate since 1990; global pneumoconiosis cases have increased globally, from 36,186 in 1990 to 60,555 in 2017. The irreversibility of lung damage and its debilitating nature have made it one of the most important occupational diseases . In China alone, pneumoconiosis accounted for 90% of occupational diseases in 2018 . Common types of pneumoconiosis include asbestosis, silicosis, and coal workers' pneumoconiosis (CWP) . Section title: Introduction Educational score: 3.9550507068634033 Domain: biomedical Document type: Other Language: en CWP develops due to prolonged exposure to coal dust, leading to varied clinical presentations. On one side, patients have near‐normal lung function with few symptoms and no change in mortality rate, diagnosed as simple CWP, also known as anthracosis. On the other side, patients experience reduced lung function, symptoms such as dyspnea and chronic cough, and a higher risk of mortality rate, diagnosed as complicated CWP, also known as progressive massive fibrosis (PMF) . CWP can manifest as mediastinal lymphadenopathy, fibrosis, nodules, consolidation, or masses requiring differentiation from other malignancies or treatable conditions like tuberculosis . Section title: Introduction Educational score: 3.493436813354492 Domain: clinical Document type: Clinical case Language: en Here, we report a case of CWP in a 75‐year‐old female with a history of cured breast cancer posing a diagnostic challenge due to its presentation as a left upper lobe lung mass resembling lung cancer. Section title: Case History Educational score: 3.543164014816284 Domain: clinical Document type: Clinical case Language: en In December 2020, a 75‐year‐old female with a history of cured breast cancer, hypertension, and more than 30 years of cooking with biomass fuels in poorly ventilated kitchens was referred to the radiology department for further evaluation of an incidental mass found on the chest X‐rays. She reported left shoulder pain persisting for a year and previously was diagnosed and treated as a frozen shoulder. About 11 months after the start of treatment, she did not notice a significant improvement in her symptoms; hence, her family physician ordered a chest x‐ray based on the suspension of possible underlying cancer. She had undergone treatment for left breast cancer 25 years ago, including total mastectomy, adjuvant chemotherapy, and radiotherapy. The chest X‐rays revealed a consolidation favoring lung mass in the left upper hemithorax , and subsequently, she was referred to our hospital. She had no respiratory or constitutional symptoms such as fever, fatigue, weight loss, cough, dyspnea, hemoptysis, etc. Section title: Methods Educational score: 3.603156328201294 Domain: clinical Document type: Clinical case Language: en Physical examination revealed controlled blood pressure and other vital signs within normal range. Left shoulder movements were painful and restricted. There was a mastectomy scar on the left side of the chest. Auscultation revealed normal lung and heart sounds. No lymphadenopathy was detected. There were no other significant findings. Primary lab tests were as follows: white blood cell 6300/μL, hemoglobulin 12.7 g/dL, platelet count 327,000/μL, urea 40 mg/dL, creatinine 0.9 mg/dL, international normalized ratio (INR) 1 and partial thromboplastin time (PTT) 30 s (reference range: 30–45 s). A chest tomography (CT) scan was ordered. It showed a solid mass with dimensions of 31 x 16 x 22 mm with a spiculated edge containing calcifications in the left upper lobe of the lung. Section title: Conclusion and Results Educational score: 3.5212810039520264 Domain: clinical Document type: Clinical case Language: en A CT‐guided percutaneous core needle biopsy was planned, and the patient was transferred to the operating room. After prepping and draping, multiple nerve blockages in the third and fourth left intercostal spaces with lidocaine 2% were done, and a biopsy specimen was taken, which subsequently caused hemopneumothorax. Immediately, a chest tube was placed, and intensive fluid therapy started. 1.5 L of blood drained into the bottle in minutes. A complete blood count was requested. The patient's hemoglobin decreased from 12.7 to 10.8 g per deciliter, and his platelet count dropped from 327,000 to 242,000/μL. Two units of packed red blood cells were infused, and after stabilization, the patient was transferred to the intensive care unit (ICU). Section title: Conclusion and Results Educational score: 4.037005424499512 Domain: biomedical Document type: Study Language: en Histopathology revealed alveolar tissue with a collection of many carbon‐laden macrophages and histiocytes with anthracosis. Fibroblast cells surrounded some areas of necrosis. No atypia or mitosis was detected. Immunohistochemistry (IHC) confirmed CWP diagnosis, with positive CD68 staining and negative panCK staining in these cells. Section title: Conclusion and Results Educational score: 1.2627716064453125 Domain: clinical Document type: Clinical case Language: en After one week of conservative management, chest tubes were removed, and the patient was discharged home. The three‐month follow‐up revealed no complications. Section title: Discussion Educational score: 3.8295223712921143 Domain: biomedical Document type: Clinical case Language: en Our case highlights the diagnostic challenge posed by CWP, particularly when mimicking lung cancer. Prolonged exposure to biomass fuel in poorly ventilated settings was identified as the underlying cause, highlighting occupational health hazards. There are multiple reports and articles about CWP mimicking lung cancer . It is important to note that CWP can increase the risk of cancer itself. A nationwide study in Taiwan showed that the standardized incidence ratio was 1.12 times more in patients with CWP than in the normal population for all cancers and 1.45 times for mediastinal and lung cancers . Section title: Discussion Educational score: 4.045745849609375 Domain: biomedical Document type: Study Language: en Typical Imaging features of CWP include < 5 mm nodules or mass lesions with upper lung zone predominance. Calcifications could be seen in 10%–20% of patients on chest X‐rays and 30% of patients on CT scans. 30% of patients might show mediastinal or hilar lymphadenopathy on CT scans. In complicated form, massive fibrosis presents as large opacities on chest X‐rays. In some cases surrounding emphysematous area could be seen around the lesion on CT scans . Our case demonstrated the typical upper lung zone location with calcifications; however, these findings can also be seen in malignant lesions, along with a spiculated border, which is a feature usually associated with malignancy. Section title: Discussion Educational score: 4.100469589233398 Domain: biomedical Document type: Study Language: en Magnetic resonance imaging (MRI), especially T2‐weighted (T2W) images, seems to be promising in situations where differentiating CWP from cancer is difficult based on CT imaging, as Ogihara and colleagues observed . All lesions diagnosed as PMF consistently exhibit low signal intensity on T2W images (100% sensitivity), whereas 15 out of 16 cancerous lesions had intermediate to high signal intensity on T2W (94% specificity). Nevertheless, differentiating PMF from cancerous lesions based on MRI with contrast enhancement did not appear to be beneficial . Additionally, Zhang et al. looked at another 25 cases with a total of 39 PMF lesions. They discovered that 38 out of 39 lesions had either low or unevenly low signal intensity on T2W and Spectral Presaturation with Inversion Recovery (SPIR). The low signal intensity was compared to the soft muscle in the chest wall. However, only two of the 34 patients with lung cancers or tumors had an uneven equal‐low signal on T2W and SPIR . Section title: Discussion Educational score: 4.000274658203125 Domain: biomedical Document type: Study Language: en Another modality for differentiating between pneumoconiosis and cancerous lesions is nuclear imaging . In one study, the use of combined F‐fluorodeoxyglucose (18F‐FDG) positron emission tomography (PET) and CT helped diagnose lung cancer with a sensitivity of 92.8% and a specificity of 87.8%. This combination was better than using FDG or CT alone in terms of reducing their high false positive rates . Section title: Discussion Educational score: 3.371311664581299 Domain: biomedical Document type: Other Language: en Both MRI and FDG‐PET/CT have shown great value in differentiating pneumoconiosis from cancer. However, it is important to acknowledge that each imaging modality can produce false positive or negative results, necessitating careful interpretation. Therefore, the use of these modalities might not completely eliminate the need for a biopsy in all cases. Section title: Discussion Educational score: 2.782487154006958 Domain: clinical Document type: Clinical case Language: en As a wrap‐up for this patient's shoulder pain, it is important to consider that Pancoast tumors can cause shoulder pain due to their invasion of surrounding nerves. In contrast, pneumoconiosis lesions are typically benign. Therefore, we conclude that any shoulder pain experienced by the patient is likely due to the inadequate treatment of the frozen shoulder rather than an underlying malignancy. Section title: Discussion Educational score: 4.136708736419678 Domain: clinical Document type: Clinical case Language: en In conclusion, this case highlights the diagnostic challenges in distinguishing CWP from lung cancer, particularly when presenting as a suspicious upper lobe lung mass. In our patient, the presence of a spiculated mass with calcifications necessitated thorough investigation to rule out malignancy, ultimately leading to a CWP diagnosis confirmed by histopathology and immunohistochemistry. This underscores the importance of a comprehensive diagnostic approach, particularly in patients with overlapping risk factors, such as a history of biomass fuel exposure and prior cancer. Multidisciplinary collaboration among clinicians, radiologists, and pathologists proved essential in reaching an accurate diagnosis, underscoring that such integration is crucial for optimal patient outcomes. Enhanced awareness of CWP's atypical presentations and potential imaging mimics, including malignancy, can support early and accurate diagnosis, thereby avoiding unnecessary interventions and reducing healthcare burdens. Section title: Author Contributions Educational score: 0.9723100066184998 Domain: other Document type: Other Language: en Yeganeh Pakbaz: writing – original draft (lead); Investigation (lead); Farzan Moodi: conceptualization (lead); writing – original draft (supporting); writing – review and editing (lead). Section title: Consent Educational score: 1.1314862966537476 Domain: other Document type: Other Language: en The patient provided written consent to publish this report, following the journal's policy on patient consent. Section title: Conflicts of Interest Educational score: 0.8662789463996887 Domain: other Document type: Other Language: en The authors declare no conflicts of interest. | Review | biomedical | en | 0.999995 |
PMC11695562 | Section title: Introduction Educational score: 0.9848646521568298 Domain: other Document type: Study Language: en Considerable new efforts to discover new Paleolithic sites in Central Asia have been made in the last decade . Several projects, including our own, have now surveyed the area located in Kazakhstan immediately south of the archaeologically rich Gorny Altai , but no significant new sites have been found. Therefore, revisiting old sites that have already been studied by Soviet scholars is of paramount importance. Unfortunately, most of these lay along the Irtysh and Bukhtarma rivers in East Kazakhstan , and were flooded due to the construction of the Bukhtarma Reservoir in 1958 and many have been submerged. Because most of them were open-air sites with poor organic preservation, there is no way to reconstruct their chronology from museum collections. Bukhtarma Cave, the first documented Paleolithic cave site in Kazakhstan, constitutes a notable exception to this rule, because its faunal collections still exist. In this report, we provide a maximal possible reconstruction of the site using the available data . Fig. 1 Map showing the location of Bukhtarma Cave and nearby important Paleolithic sites Section title: Regional Setting and General Characteristics Educational score: 0.8798363208770752 Domain: other Document type: Other Language: en The cave was located on the right bank of the Bukhtarma River, a tributary of the Irtysh carving the Carboniferous limestone foothills of the southern Altai in East Kazakhstan. It was first described by Spassky and then excavated as part of the Hermitage’s East Kazakhstan Archaeological Expedition . The site was originally published as Пeщepa/Peshchera, which simply means “cave” in Russian, and could lead to confusion with many other cave sites. We therefore redubbed it here using the river’s English name (in Russian Бyxтapминcкaя or Bukhtarminskaya/Bukhtarminskai͡a ; in Kazakh (Cyrillic) Бұқтыpмa/(Latin): Buqtyrma Üñgırı)). According to Gokhman’s description, the cave was situated to the east of the now submerged eponymous village of Peshchera, about 1.5 km from the modern river channel and ca. 30 m above it . Fig. 2 Photograph of the 1953 excavation (courtesy of the East Kazakhstan Regional Museum of Local History, Öskemen, Kazakhstan) Section title: Regional Setting and General Characteristics Educational score: 1.0239698886871338 Domain: other Document type: Other Language: en Probably, the river flowed closer to the cave during prehistoric times, because Gokhman notes that a test pit 16 m from the entrance contained river cobbles found at ~ 9 m below the cave surface . The cave entrance faced west and consisted of two chambers, named “Big” and “Small,” united by a common, partially collapsed roof . The East Kazakhstan Expedition began excavating in 1950 and continued until 1954. A total surface of 130 m 2 was excavated in both chambers. A test pit laid in the larger chamber yielded no finds so we will focus on the 80 m 2 excavated in the small chamber in 1953–1954. Section title: Stratigraphy Educational score: 0.9674543738365173 Domain: other Document type: Other Language: en Based on the profile drawings and the descriptions of the layers and their thicknesses, the deepest part of the excavation, in the southwestern corner corresponding to the 1953 pit, reached 3 m, whereas the shallowest was only 0.3 (at the entrance). The sequence is as follows : modern debris and soil covered the first 0.25–0.3 m; this was followed in some places by brownish sandy loam; and, finally, by yellow loam. Fig. 3 Horizontal plan of the excavation with an annotated transversal section from squares Д-E (courtesy of the East Kazakhstan Regional Museum of Local History, Öskemen, Kazakhstan). Translation: RI Section title: Stratigraphy Educational score: 1.0161936283111572 Domain: other Document type: Other Language: en The sequence ended with two layers of river gravels. The first three layers contained all the finds, and it appears that the brownish layer only filled in the floor cavities. According to the report, the excavators were not able to distinguish cultural levels. Given the nature of the fauna, which suggests carnivore activity in the earliest part of the sequence (see the “Faunal Analysis” section), it is reasonable to assume that some of the lateral unconformities are possibly the result of carnivore den management. Section title: Stratigraphy Educational score: 0.9917861819267273 Domain: other Document type: Other Language: en Nevertheless, the excavators noted some features, such as a hearth, and the presence of fire is also attested by five burnt bones. Section title: Lithic Assemblage Educational score: 0.9838037490844727 Domain: other Document type: Other Language: en Gokhman reported a very small lithic assemblage, with a total of ca. 50 pieces recovered from the entire excavated surface over several seasons. The drawings and description are very general, but hint at the presence of Levallois and blade technology, concluding that the assemblage is probably Upper Paleolithic. We initially believed the lithic collection to have been shipped to St. Petersburg like the faunal collection, with only a few pieces left behind to feature in the vitrine in the museum at Öskemen. However, the collection in Öskemen contains 43 pieces (including those on display), and no lithic collection could be found in any of the St. Petersburg museums (Kunstkamera, Institute for the History of Material Culture). Therefore, we surmise that the entire collection probably remained in Kazakhstan. Section title: Lithic Assemblage Educational score: 1.448068380355835 Domain: other Document type: Study Language: en The artifacts obviously belong to different periods, with several pieces typical for an earlier period such as the Middle Paleolithic and others certain to belong to a much later period, probably Holocene . The earlier period is represented by seven pieces with complex scar patterns and/or facetted platforms typical of the Levallois technique, as well as sidescrapers and bifacially retouched pieces. Four blades and seven bladelets and one microblade core represent a likely Holocene part of the assemblage. Artifacts that could be from the Upper Paleolithic, such as the three endscrapers and four blades, are more difficult to place, since they occur in both Paleolithic and Holocene contexts . Fig. 4 Lithics likely representing the earlier part of the sequence. (1) Scraper with ventral retouch on Levallois flake; (2) retouched Levallois point; (3) double sidescraper on Levallois blank with secondary retouch on the ventral side; (4) Levallois point; (5) flake with bifacial retouch (pointed part is on the retouched bulb of percussion); (6) convergent scraper; (7) convergent scraper with secondary retouch on the ventral aside; (8) proximal double scraper on Levallois blade with secondary ventral retouch; (9) atypical (thick) Levallois flake with ventral retouch; (10) denticulate on atypical Levallois blank; (11) scraper on blade with bifacial retouch at the distal end; (12) convergent scraper on blade. Note the fresher secondary retouch on pieces nos. 1, 3, 11 Fig. 5 Lithics likely representing the later part of the sequence. (1) Bifacial tool; (2–5) scrapers; (6–12) bladelets; (13–14) bladelet cores Section title: Lithic Assemblage Educational score: 1.0208420753479004 Domain: other Document type: Other Language: en The pieces are made on a variety of raw materials, including shale, chert, and porphyry, which are common in the region. Several of the Levallois pieces are made on green porphyry, the only observed association between a raw material and a technique . The pieces have distinct degrees of weathering, from very heavily smoothed out , to completely fresh . The fact that Gokhman notes the absence of patina or weathering is a further indication of the presence of some water channels that may have affected some parts of the cave but not others. Fig. 6 Association between knapping technique and ( a ) degree of weathering and ( b ) type of raw material Section title: Lithic Assemblage Educational score: 1.0068045854568481 Domain: other Document type: Other Language: en Although the sample is small, the heaviest weathering is associated with the Levallois pieces . These pieces were often secondarily retouched (e.g., three inverse sidescrapers), probably at a later time, and possibly by different inhabitants of the cave. Section title: Faunal Analysis Educational score: 3.9388837814331055 Domain: biomedical Document type: Study Language: en Our analysis was conducted in January 2020 at the Zoological Institute of the Russian Academy of Sciences in St Petersburg, Russia. The faunal remains were previously identified by Vereschchagin and Mel’nikova and sorted by taxa. The remains of 29 species of birds were studied by Panteleev , but these were not in the collection we accessed in 2020. A second anatomical identification of the mammalian remains was performed during this analysis. Non-identified remains were classified following ungulate size classes . With regard to the skeletal part profiles, all identifiable specimens (including shaft fragments) were taken into account and recorded following the “element, portion, segment” method . Analyses of the bone surfaces were conducted on all the remains. Bone surfaces were observed for the taphonomic and zooarchaeological observations under a low-angled light using a hand lens with magnification 20 × . Weathering, root etching, anthropogenic, and carnivore modifications were systematically investigated . Oxide colorations of the bone cortical surfaces were also recorded. The proportion of preserved cortical surface was estimated per quartile to estimate the proportion of anthropogenic modifications. Three remains were selected for ZooMS analysis. We sampled following the Brown et al. protocols and the analysis was conducted within the ZooSCAn ZooMS platform in Novosibirsk. Section title: Results of the Zooarchaeological Analysis Educational score: 3.728749990463257 Domain: biomedical Document type: Study Language: en The mammalian faunal assemblage contains 279 remains, of which 229 could be taxonomically identified. The high identification rate can be explained by the selective sampling of the assemblage during excavation. This selection is underlined by the abundance of teeth (NISP = 50; %NISP = 18%) and the over-representation of articular extremities for the long bone (NISP = 56; %NISP long bones = 60%), a pattern that is very different from what is usually identified in unselected assemblages from Middle Paleolithic or Upper Paleolithic contexts accumulated by either humans or carnivores. Herbivores largely dominate the faunal spectra (NISP = 183; %NISP = 80%). Represented by the most common species of the late Pleistocene in the region , the carnivore guild accounts for 16% of the identified remains. One human metacarpal was also identified. Section title: Results of the Zooarchaeological Analysis Educational score: 2.2454311847686768 Domain: other Document type: Study Language: en The Bukhtarma Cave faunal spectrum contains taxa coming from different environments (mammoth step, open steppe, woodland), and different chorological contexts (Pleistocene, Holocene), and includes wild and domesticated species, proving that the assemblage results from different archaeological horizons spanning the Pleistocene and the Holocene (Table 1 ). Table 1 Faunal spectrum of Bukhtarma Cave expressed in NISP and % NISP Taxa NISP % NISP Rodent Marmot 7 3.1% Beaver 1 0.4% Carnivores Badger 1 0.4% Fox 3 1.3% Wolf 2 0.9% Cave hyena 17 7.4% Brown bear 8 3.5% Medium-size carnivore 1 0.4% Large-size carnivore 5 2.2% Herbivores Horse 69 30.1% Hemiones 4 1.7% Camel 15 6.6% Rhinoceros 6 2.6% Mammoth 2 0.9% Bovine 51 22.3% Yak 1 0.4% Sheep 8 3.5% Giant deer 1 0.4% Roe deer 6 2.6% Red deer 20 8.7% Human 1 0.4% Total identified remains 229 Non-identified Small-size ungulate 1 Medium-size ungulate 5 Large-size ungulate 21 Megafauna 19 NID 4 TOTAL 279 Section title: Results of the Zooarchaeological Analysis Educational score: 2.0636632442474365 Domain: other Document type: Study Language: en The preservation of the material is marked by the high frequency (60%) of weathering , of which the most intense stages (3 to 5) affect 30% of the remains, suggesting that the material was exposed for a long time before burial. These alterations have direct consequences on the preservation of the remains: 40% of them exhibit the destruction of more than half of their cortical surface, limiting the observation of possible human modifications . Fig. 7 a Frequency of the weathering stage on the assemblage; b frequency of the cortical surface preservation: stage 1—more than 75% preserved; stage 2—more than 50%; stage 3—more than 25%; stage 4—less than 25% Section title: Results of the Zooarchaeological Analysis Educational score: 2.080242395401001 Domain: other Document type: Study Language: en Despite this problem of preservation, carnivore modifications on the assemblage were found in abundance (NISP = 39). The carnivore impact is observable on most of the ungulate species and includes pits, crenulated edges, groove marks, and digested bones (Table 2 ). Table 2 Carnivore impact on the material compared to the number of observed remains (preservation stages 1 and 2, teeth excluded) and the total of NISP Taxa NISP with carnivore mark NISP total Brown bear 3 8 Medium-size carnivore 1 1 Large-size carnivore 3 5 Horse 10 69 Hemiones 1 4 Camel 5 15 Mammoth 1 2 Bovine 12 51 Sheep 1 8 Roe deer 1 6 Red deer 1 20 Medium-size ungulate 1 5 Large-size ungulate 3 21 Megafauna 1 5 Non-identified 1 4 Section title: Results of the Zooarchaeological Analysis Educational score: 2.9275424480438232 Domain: biomedical Document type: Study Language: en Fifteen cut marks and six calcined bones make up the human impact on the material (NISP = 21, 18% of the remains with a good preservation of the cortical surfaces (stages 1 and 2)). Human activity was focused on three ungulates species (red deer, bovine, and horse) and on brown bear. The absence of notches and percussion marks can be explained by the scarcity of ungulate long bone shaft fragment (NISP = 45) within the faunal stock (Table 3 ) . Table 3 Human impact on the material compare to the number of observed remains (preservation stages 1 and 2, teeth excluded) and the total of NISP Taxa NISP with anthropogenic marks Observable remains NISP total Brown bear 2 4 8 Horse 8 29 69 Bovine 2 21 51 Red deer 6 16 20 Fig. 8 Six examples of cut marked bones from the Bukhtarma Cave collection used for radiocarbon dating. From left to right and top to bottom: Equus , Equus , Ursus , large ungulate , Equus , 6. Equus Section title: Results of the Zooarchaeological Analysis Educational score: 1.378885269165039 Domain: other Document type: Other Language: en In addition, five remains of horse (a metacarpal), bison (an atlas, a humerus shaft fragment), and non-identified large ungulates (two long bone shaft fragments) exhibit at the same time carnivore and human marks, suggesting that during some specific moments of the site’s history, the cave was occupied sub-contemporaneously by the two taphonomic agents. Section title: Results of the Zooarchaeological Analysis Educational score: 2.0511739253997803 Domain: other Document type: Study Language: en In order to obtain the chronology of the different occupations, 12 remains, including 10 exhibiting anthropogenic and/or carnivore modifications , were selected for direct radiocarbon dating, which was carried out at the Curt Engelhorn Zentrum für Archäometrie (CEZA), Mannheim, Germany. The results are presented in Table 4 . Table 4 Results of the 14 C dating of the collection order by chronological order. Dates were calibrated using OxCal 4.4 and the IntCal2020 dataset . In blue: remains with a probable human origin; in red: remains with a probable carnivore origin; in purple: remains with a mixed origin Section title: Discussion Educational score: 1.9607282876968384 Domain: other Document type: Study Language: en Considering the relative abundance of anthropogenic modifications (cut marks) made by lithics, as well as the abundance of carnivores in the faunal spectrum and the frequency of their marks on the material, it seems that both taphonomic agents (humans and carnivores) contributed to accumulating the assemblages. The dating confirms the observation made on the faunal spectrum and the lithics and the probable existence of multiple occupations of the cave during the Upper Pleistocene. The human occupation dates back to ca. 47 ky cal. BP as attested by the cut mark on a shaft fragment of a large ungulate . People subsequently returned in the cave at least three other times during the Pleistocene and several times during the Holocene. One of these last occupations is attested by the presence of the 7.7 ky cal. BP old human metacarpal. Section title: Discussion Educational score: 1.559923529624939 Domain: other Document type: Study Language: en We tentatively associate the earliest occupation with the typologically Middle Paleolithic tools found at the site. From that point of view, these are similar to tools known from the Gornyi Altai sites . However, as the majority of these tools appear to have been secondarily recycled at a later (unknown) date, some of their morphological characteristics are mixed and may give the impression of an evolving technology . The part of the collection that seems, on typological grounds, to belong to an earlier time period is different from assemblages from contemporary nearby sites typically assigned to the Initial Upper Paleolithic (IUP), such as Ushbulaq, a recently excavated open air site in East Kazakhstan . However, at least one bifacially retouched flake shows echoes of possible Eastern Micoquian technology, which is known from Okladnikov and Chagyrskaya Caves in the Gorny Altai, grouped under the Sibiryachikha industry and linked with a possible long-distance migration of Neanderthal groups from Europe to Siberia . Section title: Discussion Educational score: 2.360011577606201 Domain: other Document type: Study Language: en The intensification of human and carnivore interaction during MIS3 previously described at a local and continental scale finds an interesting echo here. During the first part of the site’s history, between ca. 48 and 40 ky cal. BP, Bukhtarma Cave was occupied alternatively by carnivores and hominins. Rivals et al. recently concluded that the majority of the Bukhtarma Cave assemblage has no reliable cut marks and represents a carnivore den. We show that this is clearly not the case for the part of the assemblage dated to the Upper Paleolithic. Even for the earliest period, it appears that some of these carnivore occupations took place probably sub-simultaneously to the human occupation since five bones with cut marks were secondarily gnawed on by carnivores before they lost their nutritive value. Finally, the human-carnivore interaction at Bukhtarma Cave ends with the exploitation of bear during the Holocene. Section title: Conclusions Educational score: 1.1539191007614136 Domain: other Document type: Study Language: en For now, Bukhtarma Cave remains the only Paleolithic cave site in East Kazakhstan, and possibly the only one to preserve Middle Paleolithic stone tools. Our work gives a first chronological framework establishing the baseline for an occupation of the southern (Kazakh) Altai. The low density of artifacts is likely due to sporadic visits by a small population, interspersed with carnivore occupations, as has also been shown from the Siberian Altai. Over time, after ca. 40 ky, humans become the sole accumulators of the faunal remains. Unfortunately, due to the lack of precise coordinates for the artifacts, the details of human behavior cannot be reconstructed in higher resolution from the collections and the excavation documentation, but Bukhtarma Cave represents an important Late Pleistocene Paleolithic site in Kazakhstan and a reference point for further work in the region. | Study | other | en | 0.999998 |
PMC11695569 | Section title: Introduction Educational score: 4.974819183349609 Domain: biomedical Document type: Review Language: en pH changes in plant tissues during various physiological processes signal the regulation of various aspects of growth, development, and environmental responses . Ambient pH dynamics often involves in acidification or alkalinization in the apoplast, cytoplasm, and vacuole. Auxin-mediated apoplastic acidification contributes to the regulation of morphogenesis and cell expansion . Recent data indicate that auxin receptor auxin-binding protein 1 and transmembrane kinase 1 activate plasma membrane (PM) H + -ATPase through phosphorylation, which leads to cell wall acidification and promotes cell elongation and tissue growth . Cytoplasmic acidification correlates with development of root cap cells and pollen tube during self‐incompatibility . Gradual acidification contributes to the secretory pathway of intracellular compartments . By contrast, transient apoplastic alkalinization can be attributed to various stresses, such as chloride salt and drought . Furthermore, pH dynamics has a crucial effect on several aspects of plant biology, including nutrient absorption, microbial diversity, peptide–receptor perception, and plant immunity . Fig. 1 Ambient pH plays an important role in the biological processes of plants and pathogens. It affects the secretory pathway, growth, nutritional balance, immunity, abiotic stress response, and stomatal movement of plants and the success of pathogens. Intracellular substances are secreted from the endoplasmic reticulum to the trans -Golgi network (TGN) at neutral pH and then released at an acidic pH by the MVB/PVC compartment. Ambient pH also affects enzyme activity and transport during carbohydrate metabolism. ABP1 is an auxin receptor for the TMK1-based cell-surface signal. TMK1 phosphorylates and activates the plasma membrane (PM) H + -ATPase and is required for auxin-induced H. + -ATPase activation, apoplastic acidification, and cell expansion. The stability of anthocyanins depends on the environmental pH, thus affecting the color of flowers. In soil, excess ammonium concentrations lead to increased cell acidity and impairs growth. Improper soil pH often causes nutritional imbalance, disturbance of the soil microbial community, and various physiological diseases. Abiotic stress, such as drought and salt stress, can cause host apoplastic alkalinization, whereas biotic stress can induce apoplastic acidification or alkalization. Ambient pH can be disturbed by pathogens and the changed pH can also be perceived by the pathogen, which regulate the expression of related genes and enhance their own pathogenicity. In addition, stomatal closure is accompanied by apoplastic alkalinization and cytoplasmic alkalinization in response of PAMPs and ABA in guard cells, respectively. ABA, abscisic acid; ABP1, auxin-binding protein 1; MVB, multivesicular body; PAMPs, pathogen-associated molecular patterns; PM, plasma membrane; PVC, prevacuolar compartment; TMK1, transmembrane kinase 1 Section title: Introduction Educational score: 4.453373908996582 Domain: biomedical Document type: Study Language: en The modulation of host pH is a common infection strategy for most plant pathogens to improve their pathogenicity and adaptability. During their coevolution with pathogens, plants evolved pathogen-associated molecular pattern (PAMP)- (PTI) and effector-triggered immunity (ETI) for the detection of invading pathogens and subsequently activation of defense mechanisms . The PTI pathway features a transient extracellular alkalinization, and various types of ETI pathway exhibit an association with apoplastic acidification or alkalinization . Although programmed cell death (PCD) facilitates plant immunity induction through cytoplasmic acidification, vacuolar acidification remains essential for plant antiviral immunity . In addition, pathogens have evolved the capacity to disrupt host pH to improve infection . Pathogen-induced acidification and alkalinization occur during pathogen–host interactions involving fungi, bacteria, viruses, and aphids . The apoplastic acidification mediated by pathogens seems to achieve their infection goals by affecting cell wall growth and promoting degradation , while apoplastic alkalization is critical for the formation of Pseudomonas syringae -induced lesions on leaves of Phaseolus vulgaris . Fig. 2 Apoplastic pH shifts in various host plants modulated by acidophilic and alkaliphilic pathogens. pH of plant tissues increases or decreases at different time points after inoculation compared with healthy tissues, which include leaves, fruits, roots, and twigs (Table S1 in Supplemental Information online). The green and orange–red arrows indicate acidification and alkalinization Section title: Introduction Educational score: 4.143670082092285 Domain: biomedical Document type: Review Language: en PM H + -ATPases regulate intracellular and extracellular pH homeostasis and play key roles in cell physiology and plant immunity . Acidification or alkalinization occurs through the regulation of PM H + -ATPases during plant/pathogen interactions. This review focuses on recent advances linking pH dynamics and plant immunity and summarize recent findings related to the mechanisms of acidization and alkalinization associated with pathogens and the relationship of pH dynamics with PTI, stomatal immunity, ETI, PCD, Ca 2+ , and reactive oxygen species (ROS). Insights into pH dynamics may applied for the improvement plant disease resistance. Section title: Apoplastic acidification Educational score: 4.956357955932617 Domain: biomedical Document type: Study Language: en Host apoplastic acidification is attributed to the release of organic acids from acidophilic pathogens and the activation of plant PM H + -ATPases . During host acidification, oxalic acid, citric acid, malic acid, and succinic acid are secreted by Botrytis cinerea and Sclerotinia sclerotiorum , and oxalic acid and citric acid are secreted by Aspergillus niger . Phomopsis mangiferae acidifies the host by secreting gluconic acid, citric acid, malic acid, and fumaric acid , whereas citric acid, fumaric acid, and oxalic acid are secreted by Penicillium spp. . Recently, secreted fusicoccin A from Neofusicoccum parvum combines 14‐3‐3 proteins and triggers extracellular acidification via binding to PM H + -ATPases . Moreover, low pH growth condition has also been shown to activate H + -ATPase activities in Oenococcus oeni , Penicillium simplicissimun , and Saccharomyces cerevisiae as well as in several plant species . Apoplastic acidification of Arabidopsis thaliana roots is a result of the activation of the root proton pump through phosphorylation in response to the fungus Fusarium oxysporum . Further genetic analyses have shown that B . cinerea MAPK kinase (BcMkk1) negatively regulates oxalic acid biosynthesis via impeding phosphorylation of Per-Arnt-Sim (PAS) kinase BcRim15 by the Ser/Thr kinase BcSch9 . Host tissue acidification and organic acids secretion are regulated by VELVET complex in B . cinerea and PM H + -ATPases (Pma1) in Valsa mali . For aphid pathogens, the secretion of carbonic anhydrase II (CA-II) by the aphid Myzus persicae results in apoplastic acidification of tobacco leaves . However, how the host perceives acidification signals for defense responses, such as whether it enhances the secretion of alkaline substances, remains unclear. Fig. 3 Acidification and alkalinization influenced by pathogens and signal molecules. A Main pathway of apoplastic acidification involving the release of organic acids, which results from organic acid metabolism in fungal cells. Phytotoxins from pathogens also activate the PM H + -ATPase activities of the host. B Regulation of apoplastic alkalinization through the release of ammonia, PAMPs, RALFs, effectors, and phytotoxins from pathogens. Ammonia release can be attributed to the uptake of amino acids and improvement of amino acid metabolism in fungal cells and the increase in ambient pH. The secretion of chitin and sterols from fungi is another cause of host alkalinization. C Cytoplasmic Ca 2+ signals show a close relation to PCD and cytoplasmic acidification. Although apoplastic and cytoplasmic acidification can induce HR or PCD, cytoplasmic acidification is more dependent on [Ca 2+ ] cyt . D During stomatal closure, the regulation of cytoplasmic alkalinization and vacuolar acidification is influenced by ABA, [Ca 2+ ] cyt , and effectors in guard cells. ABA, abscisic acid; BAK1, BRI1-associated receptor kinase 1; FLS2, the leucine-rich repeat receptor kinases flagellin-sensitive 2; PAMPs, pathogen-associated molecular patterns; PM, plasma membrane; RALF, rapid alkalinizing factor Section title: Apoplastic alkalinization Educational score: 4.090974807739258 Domain: biomedical Document type: Study Language: en Over the past few decades, extracellular pH alkalinization has been regarded as one of the markers of plant immune response, especially in the PTI pathway. In addition to PMAPs induction, apoplastic alkalinization has been associated with the release of rapid alkalinizing factor (RALF), ammonia, chitin, sterols from the pathogens, and the inhibition of plant PM H + -ATPases by secreted metabolites . Section title: PAMPs triggered alkalinization Educational score: 4.952834606170654 Domain: biomedical Document type: Study Language: en Apoplastic alkalinization is a result of PM depolarization, ion fluxes, and the inhibition of PM H + -ATPases upon perception of PAMPs . The alkalinization as an early cellular response was found in response to PAMPs , such as flagellin 22 (flg22), elongation factor-Tu (elf18 and elf26), 13-amino acid peptide (pep13), lipopolysaccharides (LPS), and chitin , but not in response to nlp20 and elf12 . PM depolarization induced by elf18 and flg22, and flg22-dependemt PM depolarization occurs through the involvement of the FLS2 receptor with calcium-activated anion channels . The application of PAMPs (flg22 and pep13) also increases the H + /Ca 2+ influxes and the Cl − /K + effluxes . The inhibition of PM H + -ATPase by flg22 may depend on its receptor kinase FLAGELLIN-SENSING 2 (FLS2), which phosphorylates the PM H-ATPase at Ser-899, causes pump inactivation and apoplastic alkalinization . Additionally, plant elicitor peptides (peps) are perceived by their receptors (PEPRs) to cause medium alkalinization, and pep–PEPR interaction promotes immune responses . Alkalinization in turn promotes the Pep1-PEPRs binding and enhances root immunity in Arabidopsis . Chitin fragments and chitosan are also potent PAMPs and elicit PTI in many plant species. Interestingly, chitohexaose NAG6-induced medium alkalinization was shown to be strongly suppressed by the chitinase-like effector (MpChi) of Moniliophthora perniciosa in tobacco cells . This suggested that pathogens may deploy effectors to disturb the alkalinization signal during PTI. It has been determined that transient alkalinization of the apoplast may be correlated with the degree of resistance toward pathogens, and an important next step will be to elucidate how plants transmit and trigger immune responses involving alkalinization signals. Fig. 4 Apoplastic pH shift modulated by PTI and ETI. Changes in ambient pH depend on PM H + -ATPase activity, which is regulated by host recognition of PAMPs and effectors from pathogens and plant peptides (peps and RALFs). The alkalinization induced by flg22 depends on the phosphorylation and inhibition of PM H + -ATPase activity by its receptor FLAGELLIN SENSITIVE2, whereas the elf18-induced mechanism of extracellular alkalinization remains unclear. RALF-induced alkalinization involves its receptor kinase FERONIA (FER), which phosphorylates and inhibits PM H + -ATPase activity. In addition, the involvement of RALF in PTI signal translation depends on the downstream mitogen-activated protein kinase pathway. ETI includes convergent pathways related to ambient pH. Apoplastic acidification or alkalinization depends on the activation or inhibition of PM H + -ATPase during ETI, respectively. Arrows represent promotion. Black ended arrows represent suppression. Solid lines represent a verified relationship, and dotted lines represent connections that need to be characterized further. P in the blue circle indicates phosphorylation. FLS2, FLAGELLIN-SENSING 2; HR, hypersensitive response; NLR, nucleotide-binding and leucine rich repeat protein; PCD, programmed cell death; PM, plasma membrane; RALF, rapid alkalinizing factor Section title: RALFs mediated alkalinization Educational score: 4.687294960021973 Domain: biomedical Document type: Study Language: en RALFs induce the rapid alkalinization of the extracellular compartment and regulate growth, development, and immunity in plant . However, some fungi and nematodes also produce RALFs, cysteine-rich peptides that effectively trigger the alkalinization of plant tissues. F. oxysporum has been shown to secrete F-RALF and RALF-B peptides that trigger rapid extracellular alkalinization in tomato and tobacco ( Nicotiana tabacum ) , and similar results were reported in plant-nematode interactions, where MiRALF1 and MiRALF3 from the nematode ( Meloidogyne incognita ) rapidly alkalized the extracellular medium and plant roots . Although the mechanism by which pathogenic RALFs affect host H + -ATPase activity is not clear, plant RALFs-triggered alkalinization are known to inhibit H + -ATPase activity and induce H + influx . It has been shown that the interaction of RALF with FERONIA (FER), a major RALF peptide receptor, results in the phosphorylation of Ser899 of the PM H + -ATPase, thereby leading to pump inactivation . RALF peptides may, however, function via interactions with many different downstream partners, such as cell-wall-associated leucine-rich repeat (LRR) extensin (LRX), LORELEI (LRE)-like glycosylphosphatidylinositol (GPI)-anchored proteins (LLGs), and mitogen activated protein kinase (MAPK) cascades in plants . Currently, some nematode RALFs can bind to the extracellular domain of plant FER , but it is still unclear whether the RALF- FER signaling can affect the H + -ATPase activity. The depth mechanism of extracellular alkalinization induced by RALF from pathogens still needs a long way to go. Section title: Secreted metabolites triggered alkalinization Educational score: 4.462732315063477 Domain: biomedical Document type: Study Language: en An increase in extracellular pH is accompanied with ammonia accumulation and secretion from alkaliphilic pathogens attack . It requires the uptake of amino acids, the degradation of nitrogen sources, and nutrient deprivation, and the accumulation of ammonia results in the activation of fungal appressorium formation . In addition to ammonium, induction of extracellular alkalinization is also triggered by pathogen chitin fragments in tomato and sterols in Beta vulgaris , and syringolide 1, an elicitor secreted by P . syringae into soybean ( Glycine max ) callus cells . A recent study found that secreted proteins and one of its components exo-polygalacturonase (PehC) from Ralstonia solanacearum trigger extracellular alkalinization in tomato roots . Furthermore, extracellular alkalinization can also result from the inhibition of H + -ATPase activity, mediated by pathogen phytotoxins or metabolites , such as fumonisin B1 and fusaric acid from Fusarium spp. , pseudomycin A from P . syringae , and tenuazonic acid from Stemphylium loti . Section title: pH dynamics and stomatal closure Educational score: 4.728116989135742 Domain: biomedical Document type: Study Language: en Stomatal closure is under modulation by PAMPs in biotic pathways and abscisic acid (ABA) in abiotic pathways, which are associated with pH dynamics. This regulation relies on the activation or inactivation of PM H + -ATPase, vacuolar proton pump, and membrane potential. Stomatal closure is a crucial part of PTI and limits pathogen entry, whereas rapid apoplastic alkalinization is a key and early step that result in stomatal closure upon PAMP perception . flg22 treatment caused no significant change in [H + ] cyt in guard cells . However, early stomatal closure has been characterized by cytoplasmic alkalinization in response to ABA and methyl jasmonate (MeJA) . PM H + -ATPase activity performs a specific function in an ABA-dependent pathway controlling stomatal closure . The function of ABA involves the activation of BRI1-associated receptor kinase 1 to phosphorylate A. thaliana PLASMA MEMBRANE PROTON ATPASE2 at Ser-944 and activate the H + -ATPase activity; which this condition results cytoplasmic alkalinization and initiates stomatal closure followed by Ca 2+ elevation . The stomatal closure induced by ABA occur in Ca 2+ -independent and -dependent manners. Sucrose nonfermenting 1-related protein kinase 2.6 (also known as open stomata 1) not only phosphorylates and activates SLOW ANION CHANNEL-ASSOCIATED1 (SLAC1) in a Ca 2+ -independent manner but also activates four cyclic nucleotide-gated channels (CNGC5/6/9/12) to trigger [Ca 2+ ] cyt signals that consequently activate SLAC1 and SLAC1 HOMOLOGUE 3 in a Ca 2+ -dependent manner . The treatment with MeJA increased PM H + -ATPase activity in the root tips of lettuce ( Lactuca sativa ) seedlings ; however, no concrete evidence has been provided for such regulation in guard cells. Section title: pH dynamics and stomatal closure Educational score: 4.488450050354004 Domain: biomedical Document type: Study Language: en Regardless of their differences, PAMPs- and ABA-induced alkalinization showed similarities in terms of vacuolar acidification in response to flg22 and ABA in guard cells . The vacuolar proton ATPase (V-PPase)-mediated vacuolar acidification is necessary for the rapid ABA-induced stomatal closure . Notably, the effector XopAP from Xanthomonas oryzae pv. oryzicola inhibits vacuolar acidification, which leads to stomatal opening and infection . These results imply the linkage of cytoplasmic alkalinization to vacuolar acidification during signal transduction of stomatal closure in guard cells . Another similarity is that PAMPs and ABA can cause PM depolarization, which leads to stomatal closure . PM depolarization promotes stomatal closure, whereas hyperpolarization exhibits a relation to stomatal opening. In addition, stomatal closure may involve PAMPs and ABA partially sharing pH-dependent signaling pathways. Butyric acid (BTA) is a weak acid commonly used to induce moderate cytosolic acidification. This compound induces cellular acidification and can inhibit flg22- and ABA-induced stomatal closure . Section title: pH dynamics and stomatal closure Educational score: 4.4156174659729 Domain: biomedical Document type: Study Language: en Cytoplasmic alkalinization causes the activation of K + efflux and inactivation of K + influx, which modulate ion mobilization in Vicia sativa guard cells . By contrast, guard cell acidification activates K + channel in A. thaliana 1 (KAT1) and KAT1 homolog of potato (KST1) and reduces the activity of the guard cell outward rectifying K + channel (GORK) . Thus, apoplastic alkalinization maybe closely related to PAMPs, whereas cytoplasmic alkalinization may be involved with ABA in guard cells. PAMPs and ABA largely contribute to the inducement of stomatal closure via membrane depolarization and vacuolar acidification. However, these molecules may differ in the regulation of pH-dependent stomatal closure. Section title: pH dynamics and ETI Educational score: 4.739102363586426 Domain: biomedical Document type: Study Language: en Compared with apoplastic alkalinization during PTI, the types of pathogens and the R genes determine acidification or alkalinization during ETI . Apoplastic acidification during ETI depends on the activation of PM H + -ATPases for an incompatible plant–pathogen interaction. For example, the Cladosporium fulvum effector Avr5 is recognized by the R protein Cf5 in tomato cells, leading to the activation of the PM H + -ATPase and extracellular acidification . Similarly, apoplastic acidification resulting from activated PM H + -ATPase is also observed during the interaction between barley Mla3 and the AvrMla3 from powdery mildew ( B. graminis ) . The P . syringae effector AvrB targets the plant immune regulator RIN4, and AvrB and RIN4 enhance plant PM H + -ATPase activity, while AvrB enhances bacterial virulence in a RIN4-dependent manner through inducing stomatal opening mediated by jasmonate (JA) signaling in guard cells . Apoplastic alkalinization and HR mediated by different R genes have also been linked to incompatible interactions between barley ( Hordeum vulgare ) and B. graminis . Similar results were found during the interaction of P . syringae avrD and soybean Rpg4 , confirmed by the absence of HR in two rpg4 cultivars . A recent study found that PM-localized NLRs with CC domains CC A 309, a nucleotide-binding and leucine rich repeat proteins, inhibits PM H + -ATPase activity, leading to extracellular alkalinization, followed by PCD . Additionally, a RxLR effector CRISIS2 from Phytophthora capsici also induces apoplastic alkalinization by inhibiting PM H + -ATPase activity . Thus, the apoplastic alkalinization or acidification may regulate the pH-dependent self-activation and protein fold of plant resistance proteins, coreceptor of resistance proteins, or pathogen effectors . Section title: pH dynamics and PCD Educational score: 4.691710472106934 Domain: biomedical Document type: Study Language: en PM H + -ATPase activity links defense responses and PCD in plants. PCD occurs during development or under biotic and abiotic stresses. Cytoplasmic acidification exhibits a correlation with developmentally controlled PCD (dPCD) in root cap cells and pollen tube during self‐incompatibility response within a few minutes and pathogen-triggered PCD (pPCD) . This correlation has also been observed in animal cells and yeast cells . Accumulated pieces of evidence indicate the linkage of nucleotide-binding leucine-rich repeat proteins (NLRs) and effector-induced cell death to the inhibition of PM H + -ATPase activity . The PM H + -ATPase activator fusicoccin compromise several NLR-induced cell deaths . By contrast, the overexpression of bacterial H + -ATPase in tobacco plants triggers a hypersensitive response (HR)-like PCD . In addition, a low-pH buffer and PM H + -ATPase activation increase the number of cells exhibiting HR . Moreover, pathogens secrete organic acids that not only cause extracellular acidification but also induce cell death ; this finding suggests the promotion of pPCD is intensified by the activation of PM H + -ATPase and extracellular acidification. Such a contradiction may be attributed to the disturbance of PM potential by PM H + -ATPase and pH shift, which lead to an imbalance in ion fluxes during pPCD . The PM potential in healthy cells is coupled with the regulation of ion channels and transporters; however, the loss of PM potential shows an association with HR-like death . Section title: pH dynamics and PCD Educational score: 4.845063209533691 Domain: biomedical Document type: Study Language: en Among various ion fluxes, cytoplasmic Ca 2+ signals bear a close relation to pPCD. In the presence of flg22, Ca 2+ efflux increases the level of external Ca 2+ ; then, Ca 2+ reaches the cytoplasm through cyclic nucleotide gated channels (CNGCs), which elevates the level [Ca 2+ ] cyt sensed by calcineurin B-likes (CBL) 2/3 and CBL-interacting protein kinases (CIPK) 3/9/36. The subsequent phosphorylation and activation of vacuolar Ca 2+ /H + exchangers (CAX1/3) under the action of these Ca 2+ -sensor kinases enable the transport of excess Ca 2+ to the vacuole and release of H + into the cytoplasm . CCHA1 is a chloroplast-localized potential Ca 2+ /H + antiporter in A . thaliana , and cytoplasmic pH in ccha1 mutant is higher than in the wild-type . The findings suggest the possible relation of cytoplasmic acidification to Ca 2+ -dependent CAX activation . The shape of cytoplasmic Ca 2+ signal depends on the balance among CNGC-mediated Ca 2+ influx, PM-, endoplasmic reticulum (ER)- mediated efflux, and tonoplast-localized Ca 2+ pumps . CNGC2 and CNGC4 contribute to the induction of HR . Knockout of vacuolar Ca 2+ pumps ACA4 and ACA11 reveal salicylic acid (SA)-dependent PCD in Arabidopsis leaves during pathogen attack . Silencing of ER-localized Ca 2+ -ATPase ( NbCA1 ) accelerates the PCD induced by pathogens . These data imply that pPCD is an outcome of [Ca 2+ ] cyt elevation, Ca 2+ sensing, and Ca 2+ signal transduction . Furthermore, pPCD may be linked to the nuclear Ca 2+ ([Ca 2+ ] nuc ), which is generated by Ca 2+ influx from the cytoplasm . Ca 2+ -dependent nuclease has been shown to participate in nuclear DNA degradation during dPCD in Citrus fruits but not pPCD. Nuclear-membrane localized CNGCs (CNGC15a/b/c) and ER-localized Ca 2+ -ATPase (NbCA1) account for nuclear calcium oscillation; their actions cause alterations in the chromatin state and regulates transcriptional reprogramming . The relationship between [Ca 2+ ] nuc signature and pPCD still needs further investigation. Section title: pH dynamics and Ca 2+ Educational score: 4.676987171173096 Domain: biomedical Document type: Study Language: en Cell types and signaling molecules determine the type of relationship between pH dynamics and [Ca 2+ ] cyt . Upon flg22, stomatal closure occurs with apoplastic alkalinization and [Ca 2+ ] cyt elevation; however, [H + ] cyt in guard cells showed no significant change . By contrast, flg22 functions in the inducement of [Ca 2+ ] cyt elevation and cytosolic acidification in mesophyll cells . The stomatal closure in guard cells induced by ABA is accompanied with rapid cytosolic alkalinization followed by [Ca 2+ ] cyt elevation . In leaf and root cells, increases in [Ca 2+ ] cyt are accompanied with cytosolic acidification in response to external stimuli . BTA induces cytosolic acidification, which reduces the [Ca 2+ ] cyt levels in guard cells, whereas BTA potentiates [Ca 2+ ] cyt in mesophyll cells . The difference among various cell types can be attributed to the pH sensitivity of Ca 2+ channels. Extracellular K + and anion fluxes affect cellular Ca 2+ and H + , which cause shifts in cytosolic and vacuolar pH and changes in [Ca 2+ ] cyt and membrane potential . In addition, extracellular Ca 2+ closes stomata, causes transient oscillatory cytosolic alkalinization, and increases [Ca 2+ ] cyt in guard cells . However, organic acids from pathogens, such as oxalic acid and citric acid, can chelate Ca 2+ and reduce its activity . The chelation of extracellular Ca 2+ results in the reduced amplitude of [Ca 2+ ] cyt elevation and cytosolic acidification in root tip cells . Therefore, these findings suggest that various signaling molecules that affect [Ca 2+ ] cyt and [H + ] cyt dynamics are linked to cell types, ion channels, and membrane potential. Section title: pH dynamics and Ca 2+ Educational score: 4.254049301147461 Domain: biomedical Document type: Study Language: en During pathogen infection, [Ca 2+ ] cyt performs certain functions in PTI and ETI . However, the connection of [Ca 2+ ] cyt and pH dynamics in both types of immunity remains incompletely understood. ABA- and PAMP-induced stomatal closures lead to an increase in [Ca 2+ ] cyt . Regardless, further exploration is needed to determine whether their shared pH-dependent mechanisms trigger stomatal closure. In addition to guard cells, [Ca 2+ ] cyt elevation shows a close association with cytosolic acidification during pPCD and dPCD . Thus, pPCD and dPCD may occur beyond a certain threshold of [Ca 2+ ] cyt . Section title: pH dynamics and ROS Educational score: 4.556928634643555 Domain: biomedical Document type: Study Language: en Alkalinization has been linked to ROS burst in host cells during PTI. ROS elevation occurred in tomato and N . benthamiana leaves induced by F . oxysporum rapid alkalinizing factor (RALF)-B and in tomato roots induced by PehC from Ralstonia solanacearum during extracellular alkalinization . Apoplastic ROS production depends on nicotinamide adenine dinucleotide phosphate oxidases (NOXs or respiratory burst oxidase homologs (RBOHs), cell wall peroxidases (PRXs), and amine oxidases . Extracellular alkalinization is a prerequisite for PRX-mediated extracellular ROS production . Furthermore, the activation of PM H + -ATPase causes reduction in the ROS burst in response to flg22 ; thus alkalinization may be a crucial component of ROS signaling. Interestingly, RBOHF also participates in PAMP-induced apoplastic alkalinization in Arabidopsis guard cells . Cytoplasmic alkalinization in guard cells precedes ROS production during MeJA- and ABA-induced stomatal closures ; this finding indicates the presence of a mutually reinforcing relationship between alkalinization and ROS production. Section title: pH dynamics and ROS Educational score: 4.174294948577881 Domain: biomedical Document type: Study Language: en Extracellular signals also influence pH dynamics and ROS. The application of 20 and 200 µM H 2 O 2 strengthened cytosol acidification within 10–20 min in guard cells, but stomatal aperture was unaltered in Nicotiana tabacum . However, recent data show that the application of 10 and 100 µM H 2 O 2 effectively induced stomatal closure in Arabidopsis . Such a contradiction in the results may be attributed to the sensitivity of various plants to H 2 O 2 . Conversely, acidic extracellular pH suppressed the flg22-triggered cytoplasmic ROS burst in Arabidopsis seedlings . Therefore, the importance of cytoplasmic or apoplastic ROS for stomatal closure should be explored in detail. Section title: pH dynamics and ROS Educational score: 4.427425384521484 Domain: biomedical Document type: Study Language: en ROS elevation in cytoplasm is closely associated with cytoplasmic acidification during dPCD . Chloroplastic ROS contribute to ETI-induced pPCD in response to pathogens; cytoplasmic acidification also occurs during pPCD , but knowledge on its correlation with the pPCD process compared with dPCD is limited. Thus, further studies are required to determine whether cytoplasmic acidification triggers the accumulation of cytoplasmic ROS during pPCD. Although the accumulation of cytoplasmic ROS partially depends on apoplastic ROS transport through aquaporin PIP2;1, cytoplasmic ROS are largely generated as metabolic by-products from chloroplasts and mitochondria . An increasing number of reports indicate that cytosolic oxidation is independent of RBOHD-dependent apoplastic oxidation events for stomatal immunity . In addition, no clear distinction has been observed between intracellular and extracellular ROS or the various ROS types in numerous experimental systems. The relationship between pH dynamics and ROS requires further exploration through the integration of ROS compartmentalization and accurate measurement of the types of ROS . Section title: Concluding remarks Educational score: 4.464841842651367 Domain: biomedical Document type: Study Language: en The perception and response to pH shifts is crucial for plant survival under biotic and abiotic stresses. In response to apoplastic acidification induced by aphids, plants expedited vesicle trafficking to augment pectin transportation and fortify the cell wall . Under flooding conditions, SLAH3 S-type anion channels serve as intracellular pH sensors that perceive cytosolic acidification and promote membrane depolarization in A. thaliana roots . Under alkaline stress, alkaline tolerance 1 negatively modulates the phosphorylation level of PIP2s, which reduces their H 2 O 2 export activity under alkaline stress. This condition leads to the overaccumulation of H 2 O 2 and reduced tolerance of crops to alkaline stress . However, whether a similar mechanism exists in pathogen-mediated alkalinization remains unclear. As extracellular pH sensors, peptides (root meristem growth factor 1 and Pep1) and their receptors (RGFR and PEPR) regulate extracellular pH-mediated growth and immunity in A. thaliana roots . Regardless of the increase in the number of identified pH sensors, other cell type-specific pH sensors that sense pH shifts mediated by pathogens or abiotic stresses must be discovered. Such goals may be achieved with the assistance of large-scale and high-resolution single-cell multiomics analysis combined with live measurement of pH in specific cell types . Section title: Concluding remarks Educational score: 4.180324077606201 Domain: biomedical Document type: Study Language: en The shift in pH varies between compatible and incompatible cultivars. Activation of PM H + -ATPase by Sclerospora graminicola infection occurs in all resistant cultivars of pearl millet but not in susceptible cultivars ; this condition suggests the correlation of the level of H + -ATPase with the degree of resistance. Conversely, the interaction of the alkaline pathogen P . syringae with Phaseolus vulgaris resulted in the higher degree of alkalinization caused by incompatible pathogens than that caused by compatible pathogens . Thus, the degree of alkalinization relates to microbial pathogenicity and defense response of the host. Further, scholars should focus on cultivar-specific differences in pH perception and regulation of ambient pH. Section title: Concluding remarks Educational score: 4.169766426086426 Domain: biomedical Document type: Review Language: en Given the acid-producing characteristic of pathogens, pH-sensitive fungicides can be used for the precise control of plant diseases based on pH-responsive delivery systems . PM H + -ATPase, RALFs, and pH signaling transcription factor PacC not only modulate the ambient pH of the host but also regulate pathogen development and pathogenicity . Therefore, plant disease resistance may be improved by silencing these genes via host- or spray-induced gene silencing . The successfully designed pH-responsive proteins can be assembled and depolymerized based on pH shifts . The release of disease-resistant proteins that target specific pH environments will contribute to disease prevention and control. In summary, pH dynamics reflect an ongoing evolutionary interaction linked to various aspects of plant pathosystems. Despite the recent advances and crucial and new insights, significant gaps in our knowledge remain. Answering these questions will contribute to the development of innovative strategies for the enhancement of plant immunity and disease resistance. Section title: Supplementary Information Educational score: 1.4485911130905151 Domain: biomedical Document type: Other Language: en Supplementary Material 1. | Review | biomedical | en | 0.999997 |
PMC11695638 | Section title: 1 Introduction Educational score: 3.941910982131958 Domain: biomedical Document type: Review Language: en Thrombotic events and their complications, such as acute myocardial infarction, pulmonary embolism, and acute ischemic stroke (AIS), are leading causes of death globally and are major contributors to the global health burden . Restoration of blood flow and improvement of perfusion to the affected organ are essential for survival. Therefore, the mainstay of treatment has focused on the breakdown of the clot through thrombolysis with a tissue plasminogen activator . Section title: 1 Introduction Educational score: 4.486074924468994 Domain: biomedical Document type: Review Language: en The efficient formation of fibrin is critical for hemostasis and wound healing; however, if too much fibrin is produced and/or is deposited in non-bleeding locations, debilitating blood clots may occur. Clot structure is dependent on the properties of individual fibrin fibers, which come together to form a fibrin mesh. Clots differ in their structure in terms of fibrin fiber diameter, density, stiffness, and permeability. Fibrinolysis, specifically the breakdown of fibrin elements of a clot, is the proteolytic degradation of fibrin to facilitate the dissolution and clearance of blood clots from circulation . The serine protease tissue plasminogen activator (t-PA), a physiologically occurring glycoprotein, is the main endogenous mediator of fibrinolysis . A recombinant version, rt-PA (alteplase), was the standard fibrinolysis treatment in acute myocardial infarction, pulmonary embolism, and AIS. Tenecteplase has been modified from alteplase at three positions, resulting in an increased half-life, higher fibrin selectivity, and increased resistance to plasminogen activator inhibitor-1 (PAI-1) . Therefore, tenecteplase can be administered as a single-dose bolus application during the early stages of an acute myocardial infarction . Tenecteplase is licensed for the treatment of ST-elevation myocardial infarction (STEMI), a life-threatening emergency resulting from complete occlusion of a coronary artery by a clot, and for the fibrinolytic treatment of AIS . Several studies have reported on higher reperfusion rates, similar safety and efficacy, and improved treatment times for tenecteplase in comparison with alteplase . Section title: 1 Introduction Educational score: 4.358877182006836 Domain: biomedical Document type: Study Language: en Fibrinolysis and its regulatory mechanisms involve a complex system of biochemical reactions . The activity of tenecteplase depends on fibrin binding and, for efficient conversion of plasminogen to plasmin and subsequent plasmin-driven clot lysis, tenecteplase needs to enter a clot containing fibrin and plasminogen. After activation of plasminogen to plasmin by tenecteplase, plasmin further increases the fibrinolytic activity of tenecteplase by cleaving the single-chain into the two-chain form of the molecule . Section title: 1 Introduction Educational score: 4.228528022766113 Domain: biomedical Document type: Review Language: en It is important to note that several products containing tenecteplase exist. Whilst generic versions of new chemical entities contain the same chemical substance as the original drug (and therefore elicit the same quality and performance characteristics), biologic drugs, also known as biopharmaceuticals, are inherently more complex molecules. This is due to several factors, including structural intricacy and variability, formulation, sensitivity, and immunogenicity. Moreover, the manufacturing process of biologic drugs can significantly influence their properties. For development and production, it is crucial to consider general quality attributes that can significantly influence the performance and safety of biopharmaceuticals. These attributes encompass a wide range of factors, including post-translational modifications, molecule-related impurities, process-related impurities (PRIs), and other product-specific attributes. In the context of PRIs, the presence and quantity of host cell proteins (HCPs) in a biopharmaceutical product can have significant implications for its safety, efficacy, and overall quality. High levels of HCPs can potentially lead to adverse immune responses in patients, impacting the drug’s safety profile. Additionally, certain HCPs may interact with the drug substance, potentially affecting its stability, potency, and overall performance . Therefore, the production of biologic drugs requires meticulous attention to ensure consistency, efficacy, and safety . Section title: 1 Introduction Educational score: 1.600986361503601 Domain: biomedical Document type: Other Language: en Copy versions of tenecteplase include Mingfule ® in China, which was approved by China’s National Medical Products Administration in 2015 for use in patients with STEMI, and is also approved for AIS . Section title: 1 Introduction Educational score: 4.076383113861084 Domain: biomedical Document type: Study Language: en This study aimed to compare the fibrinolytic activity and overall product quality of the original tenecteplase, Metalyse ® 25 mg/vial presentation (Boehringer Ingelheim Pharma GmbH and Co. KG, Ingelheim, Germany), to the 16 mg/vial formulation of the tenecteplase copy Mingfule ® (CSPC Recomgen Pharmaceutical, Guangzhou, Co., Ltd.). Section title: 2 Materials and methods Educational score: 4.166121482849121 Domain: biomedical Document type: Study Language: en All release testing was performed using International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use validated methods according to standard operating procedures, which are applied to the routine quality control testing of tenecteplase for market release of batches produced by Boehringer Ingelheim. An overview of intermediate precision of methods is given in Table 1 . Testing was supplemented by state-of-the-art mass spectrometry (MS) analysis and surface plasmon resonance (SPR) measurements (an optical biosensing technique to study molecular interactions in real time). Furthermore, HCP quantification and clot lysis testing following plasmin incubation over time were performed. Section title: 2.1 Drugs to be investigated Educational score: 4.092214584350586 Domain: biomedical Document type: Study Language: en Three batches each of Metalyse ® 25 mg/vial and Mingfule ® 16 mg/vial (exclusively available in China) were analyzed. Both drugs were stored at the recommended storage conditions and were well within the expiration date (7–8 months for Metalyse ® batches, and 8–13 months for Mingfule ® batches). Following the respective instructions for use, both drugs were reconstituted with water for injection, yielding solutions with concentrations of 5.1 mg/mL for Metalyse ® and 5.4 mg/mL for Mingfule ® . Samples were reconstituted in the same time frame and stored at −20°C until analytical testing was performed. The analytical testing was conducted based on the actual protein concentration in each solution – i.e., the same amount of protein was used allowing a direct comparison of results between Metalyse ® and Mingfule ® . Section title: 2.2.1 Preparation of reagents Educational score: 4.11338472366333 Domain: biomedical Document type: Study Language: en Thrombin was reconstituted in 1 mL G-water (molecular grade water) and on the day of analysis diluted to 33 U/mL in assay buffer . Fibrinogen was reconstituted in assay buffer to 2 mg/mL, incubated at 37°C with infrequent gentle mixing to facilitate dissolution, and filtered through a pleated filter. Plasminogen was reconstituted in G-water to a final concentration of 1.5–1.9 mg/mL. Each clot lysis assay included freshly prepared reference standard and a positive control sample of the current material for release testing. Section title: 2.2.2 Clot lysis analysis Educational score: 3.4989283084869385 Domain: biomedical Document type: Study Language: en In vitro clot lysis activity of Mingfule ® and Metalyse ® samples was determined using an automated analysis system as described before . Section title: 2.2.2 Clot lysis analysis Educational score: 4.137026786804199 Domain: biomedical Document type: Study Language: en Plasmin-induced potency activation of Metalyse ® and Mingfule ® was carried out using plasmin , which was freshly diluted at a ratio of 1:25 in G-water prior to use. For the plasmin-induced conversion of single-chain to two-chain form, followed by clot lysis analysis, the Metalyse ® or Mingfule ® samples were washed with the Metalyse ® formulation buffer using 30 kDa Amicon centrifuge filters. A 500 µL sample volume (5 mg/mL) was mixed with 2 µL of human plasmin (concentration: 2.32 mg/mL; specific activity 22.6 U/mgP) and incubated at 37°C and 300 rpm on a shaking heater for a maximum of 6 h. At multiple time points, 20 µL of each sample was mixed with 780 µL dithiothreitol (DTT) to stop the enzymatic conversion. The Metalyse ® and Mingfule ® samples were denatured by incubating at 80°C for 8 min. For each measurement time point, 20 µL of the reaction mixture was diluted in assay buffer to a concentration of 32 μg/mL and subsequently analyzed using the ACL TOP ® system according to the published standard procedure. The effect of plasmin on the clot lysis activity was assessed. No clot lysis activity was observed in the presence of plasmin but absence of Metalyse ® /Mingfule ® . Section title: 2.3.1 Preparation of reagents Educational score: 4.162245273590088 Domain: biomedical Document type: Study Language: en Fibrinogen from human plasma was reconstituted in sodium phosphate buffer (0.06 M, pH 7.4) to a final protein concentration of 2 mg/mL. The solution was incubated at 37°C for approximately 30 min until the solution was completely clear. The solution was filtered through a pleated filter to remove possible precipitates and stored at 2°C–8°C or on ice until use. Thrombin from human plasma was dissolved in 1 mL purified water and diluted to 60 U/mL in HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.05% Tween 20; pH 7.4), termed Component A. Coagulation factor XIII from human plasma was diluted in Ca-HBS (HBS-EP+ mixed 1:1 with 4 M CaCl 2 [Carl Roth, cat #CN93.2]) to a final protein concentration of 50 μg/mL, termed Component B. Solutions of fibrinogen, thrombin, and factor XIII were freshly prepared and used the same day. Section title: 2.3.2 Preparation of a ready-to-use SPR-Biosensor chip and binding analysis Educational score: 2.7332870960235596 Domain: biomedical Document type: Study Language: en All SPR-based measurements were determined using a Biacore T200 analytical system (Cytiva) with the analysis temperature set to 25°C. SPR system and dilution buffer HBS-EP+ and SPR-Biosensor chips were purchased from Cytiva. Section title: 2.3.2 Preparation of a ready-to-use SPR-Biosensor chip and binding analysis Educational score: 4.210487365722656 Domain: biomedical Document type: Study Language: en Briefly, fibrinogen was immobilized on the sensor chip surface using (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide, hydrochloride)/N-hydroxysuccinimide (EDC/NHS) amine coupling . For this, fibrinogen was diluted in 10 mM sodium acetate, pH 5.5, to a final protein concentration of 100 μg/mL. An immobilization level of 12,000 resonance units (RU) was targeted by applying the immobilization wizard (aim for immobilized level) of the Biacore Control software. Unreacted, activated carboxyl groups were subsequently deactivated using ethanolamine. The immobilized fibrinogen was then cross-linked by 20 injections of a 1:1 mixture of Components A (thrombin) and B (coagulation factor XIII). Injections of this mixture were performed at a flow rate of 5 μL/min and with a contact time of 360 s. After the last injection, the matrix was conditioned with 1x HBS-EP+ at a flow rate of 50 μL/min for 3 h. A blank immobilization using EDC/NHS and subsequent deactivation with ethanolamine was performed to prepare the reference flow cell. Section title: 2.3.2 Preparation of a ready-to-use SPR-Biosensor chip and binding analysis Educational score: 4.109609603881836 Domain: biomedical Document type: Study Language: en Before measuring a sample, the matrix was conditioned using five injections of 10 mM HCl (regeneration solution) at a flow rate of 30 μL/min, a contact time of 24 s each, and a stabilization time of 60 s after each injection. During the assay run, each injection was performed with a flow rate of 5 μL/min, with a contact time and dissociation time of 120 s each. Regeneration of the chip involved three injections of 10 mM HCl at a flow rate of 30 μL/min and a contact time of 24 s each, as well as a final stabilization time of 120 s. Section title: 2.3.2 Preparation of a ready-to-use SPR-Biosensor chip and binding analysis Educational score: 4.1232686042785645 Domain: biomedical Document type: Study Language: en Binding curves were double-referenced by first subtracting nonspecific binding from a reference flow cell and then subtracting a blank cycle where buffer was injected instead of protein sample. These were then analyzed using the sensorgram comparison module of the Biacore T200 evaluation software (ver. 3.2) as described earlier . Three different Metalyse ® lots, each measured in duplicate and in three concentrations (2, 3, and 4 μg/mL), were used to define a comparison corridor (average ± 3 standard deviation [SD] approach with normalized sensorgrams). Mingfule ® lots, measured under identical conditions, were then compared (association and dissociation) to this corridor, yielding a similarity score . Section title: 2.4.1 Chinese hamster ovary cell protein (CHOP)-enzyme-linked immunosorbent assay (ELISA) Educational score: 4.114998817443848 Domain: biomedical Document type: Study Language: en The quantification of the HCP CHOP was performed by two ELISA methods using polyclonal CHOP antibodies. The Metalyse ® -specific assay (goat anti-CHOP, prepared in-house) and a commercially available assay (CHO HCP ELISA Kit, 3G [F550-1], Cygnus) were utilized. All assays included spike and assay controls, and a polyclonal, horseradish peroxidase-coupled antibody was used with fluorescence detection of absorption at 450 nm and reference signal at 630 nm. Fluorescence signals were converted to units, with 1 U corresponding to 1 ng of CHOP standard. Section title: 2.4.2 MS-based HCP analysis Educational score: 4.470584869384766 Domain: biomedical Document type: Study Language: en The MS-based HCP analysis was performed by subjecting samples to shotgun tandem mass spectra (MS/MS) analysis for unbiased protein identification, with the proteins being diluted, digested, and separated based on hydrophobicity before being subjected to the mass spectrometer. Interfering buffer components were removed through precipitation with trichloroacetic acid, washing with acetone, and resuspension in 8 M urea buffer. The samples were then reduced, carbamidomethylated, and proteolytically cleaved using trypsin. HPLC-ESI-MS and -MS/MS mass spectra were obtained using a UHPLC system coupled to an Orbitrap mass spectrometer, with peptides separated using a specific gradient and recorded in positive ion mode. The trap column was a reversed-phase (RP) chromatography column with 0.5 cm and an inner diameter of 300 μm. The separator column used was an EASY Spray PepMap Neo column (75 μm × 500 mm, 2 μm, 100 Å pore size, Thermo Fisher Scientific). Eluents were A: water/0.1% formic acid; B: acetonitrile/0.1% formic acid. The peptides were separated using a gradient that started from 2% B to 3% B in 2 min, then from 3% B to 23% B in 45 min, and finally from 23% B to 45% B in 15 min at 45°C with a flow rate of 0.25 μL/min. The eluted peptides from the analytical column were subjected to positive ionization at 1.5 kV using the EASY-Spray™ Source (ThermoFisher Scientific, Waltham, MA, United States) of an Exploris 480 mass spectrometer. The mass spectrometer was operated in data dependent acquisition (DDA) mode with survey scans acquired from m/z 375 to 1,200 in the Orbitrap analyzer at a resolution of 120,000, followed by frag mentation of the 20 most abundant ions. MS/MS were obtained using higher-energy collisional dissociation (HCD) at 30%. The isolation window was set at 2 m/z, the Orbitrap resolution at 15,000, the target value at 5E4, and the maximum injection time set at auto. Selected precursor ions for fragmentation (including charge state 2–5) were excluded for 45 s, and the repeat count was set at 1. Protein identification and quantification were achieved using Proteome Discoverer 3.0 and Progenesis QI for Proteomics, respectively, with a false discovery rate below 0.05 on peptide spectrum match and 0.01 on protein level, and only proteins identified in all three technical replicates were quantified. Section title: 2.4.3 Monomer content quantification Educational score: 4.132298946380615 Domain: biomedical Document type: Study Language: en The monomer and aggregate content were determined using high-performance size exclusion chromatography (HP-SEC). HP-SEC separates molecules according to their molecular size on a SEC column with a porous column matrix (Tosoh Bioscience). Larger molecules have a shorter residence time in the pores and elute first from the column, whereas smaller molecules are seen in the chromatograms as peaks with a greater retention time as they elute later. With this method, the monomers can be separated from high-molecular-weight species (HMW = aggregates). For analysis, an isopropanol/L-arginine/ammonium sulfate buffer at pH 7.3 was used as mobile phase with an isocratic flow of 0.5 mL/min. Analysis was monitored at 280 nm. The relative monomer and aggregate content were calculated as a percentage of the total peak area values. Section title: 2.4.4 Single-chain determination by HP-SEC Educational score: 4.172399520874023 Domain: biomedical Document type: Study Language: en The percentage of the single-chain variant in the samples was determined by HP-SEC under reducing conditions. The tenecteplase molecule exists as single- and two-chain variants due to an internal clipping site between amino acids arginine 275 and isoleucine 276. The two resulting variants of the molecule are linked by disulfide bridges that are reduced by the addition of DTT. Due to the differences in the molecular weight, single- and two-chain variants can be separated by HP-SEC using a SEC column with a porous column matrix (Tosoh Bioscience). For analysis a sodium dihydrogen phosphate monohydrate/sodium dodecyl sulfate buffer at pH 6.8 was used as mobile phase with an isocratic flow of 0.6 mL/min. Samples were reduced by mixing with DTT and incubated at +80°C for 8 min. Analysis was monitored at 214 nm. The relative content of the single-chain variant was calculated as a percentage of the total peak area values. Section title: 2.5.1 Glycosylation analysis using MS Educational score: 4.185815811157227 Domain: biomedical Document type: Study Language: en MS-based analysis was used to determine the relative abundance of N- and O-glycans. Samples were denatured in guanidinium hydrochloride, reduced using dithiothreitol, alkylated iodoacetic acid, and finally digested using trypsin at pH 7.4. The resulting peptides were subjected to C18 (XSelect Peptide CSH C18 Column, Waters) reversed-phase ultra-performance liquid chromatography (RP-UPLC, Acquity, Waters) coupled to high-resolution MS (Q-Exactive, Thermo Scientific) including high-energy collision-induced fragmentation. Fragmentation data and accurate precursor mass were used to identify glycopeptides. For data processing (Byos V4.2, Protein Metrics Inc.), selected ion chromatograms (SICs) of all relevant N- and O-glycopeptides were generated. Relative site-specific quantitation was conducted for each respective glycan by dividing the relative SIC area of the glycan by the sum of SIC areas of all glycans at the specific glycosylation site. Section title: 2.5.2 N-glycosylation occupancy: Type I/II determination Educational score: 4.1729559898376465 Domain: biomedical Document type: Study Language: en Two major N-glycosylation variants of tenecteplase are present: type I and type II. Type I is glycosylated at asparagine (Asn) positions Asn103, Asn184, and Asn448, whereas type II is glycosylated at positions Asn103 and Asn448. For the determination of type I and type II content, samples were cleaved enzymatically by plasminogen, reduced with DTT, and separated by RP-HPLC using a C8 column (Agilent). Eluent A (0.1% trifluoroacetic acid in water) and eluent B (0.1% trifluoroacetic acid in acetonitrile) were used as mobile phases. For elution, a gradient with increasing acetonitrile content (20%–37% eluent B) in the mobile phase at a flow rate of 1 mL/min was applied. Quantitation of type I and type II variants was performed by ultraviolet detection at 214 nm with area percentage evaluation. Section title: 2.6.1 SPR clearance receptor binding assay (LRP-1, ASGR, MR) Educational score: 4.166948318481445 Domain: biomedical Document type: Study Language: en Recombinant low-density lipoprotein receptor-related protein 1 (LRP-1) Cluster IV , asialoglycoprotein receptor 1 , and mannose receptor C, type 1 were reconstituted according to the manufacturers’ instructions. CaCl 2 was purchased from Merck . All SPR measurements were performed using a Biacore T200 (Cytiva) with the analysis temperature set to 25°C. SPR running and dilution buffer HBS-EP+ and SPR-Biosensor chips were purchased from Cytiva. The standard running buffer HBS-EP+ was supplemented with 8 mM CaCl 2 if not otherwise specified. Prior to immobilization, all receptors were diluted in 10 mM sodium acetate buffer (pH 4.5) to a final protein concentration of 35 nM. LRP-1 , ASGR1 , and MRC1 (approx. 13,000 RU) were then immobilized on parallel flow cells using the EDC/NHS amine coupling kit . Unreacted, activated carboxyl groups were subsequently deactivated using ethanolamine. For preparation of the reference flow cell, a blank immobilization using EDC/NHS and subsequent deactivation with ethanolamine were performed. Section title: 2.6.1 SPR clearance receptor binding assay (LRP-1, ASGR, MR) Educational score: 4.095796585083008 Domain: biomedical Document type: Study Language: en For interaction analysis, Metalyse ® (88, 53, 32 μg/mL) and Mingfule ® (53, 32, 19 μg/mL) were injected over all flow cells (flow cells 1–4) at a flow rate of 30 μL/min for 240 s, following a dissociation time of 150 s. The surface was then regenerated using a 120 s injection of HBS-EP+ at 30 μL/min (without CaCl 2 ). Before each assay run, the matrix was conditioned using 15 injections of reference standard material at 88 μg/mL. Binding curves were analyzed as described in section 2.2 . Section title: 2.7.1 Analysis of post-translational modifications Educational score: 4.270747184753418 Domain: biomedical Document type: Study Language: en Post-translational modifications, including methionine and tryptophan oxidation, asparagine and glutamine deamidation, or lysine glycation, were assessed using MS-based analysis. Samples were denatured in urea, reduced using tris(2-carboxyethyl) phosphinehydrochlorid, and alkylated using iodoacetic acid. Digestion using trypsin was carried out at pH 6.0 for succinimide and isoaspartate analysis, and at pH 7.4 for analysis of all other post-translational modifications. Samples were additionally de-N-glycosylated using glycosidase F. The resulting peptides were subjected to C18 (XSelect Peptide CSH C18 Column, Waters) RP-UPLC (Acquity, Waters) coupled to high-resolution MS including high-energy collision-induced fragmentation (Q-Exactive, Thermo Scientific). Fragmentation data and accurate precursor mass were used to identify unmodified and modified peptides. For data processing, SICs of all relevant modified and unmodified peptides were generated (Byos V4.5-53, Protein Metrics Inc.). Relative site- or peptide-specific quantitation was conducted for each modification by dividing the relative SIC area of the respective modified peptide by the sum of SIC areas of the respective modified and unmodified peptides. Section title: 2.7.1 Analysis of post-translational modifications Educational score: 4.201401710510254 Domain: biomedical Document type: Study Language: en MS-based analysis was used to determine the relative abundance of thiols in the samples. The samples were denatured in guanidine hydrochloride and labeled with N-ethylmaleimide (NEM), after which they were reduced using dithiothreitol, alkylated using iodoacetic acid (IAA), and finally digested using trypsin. The resulting peptides were subjected to C18 (XSelect Peptide CSH C18 Column, Waters) RP-UPLC coupled to high-resolution MS including high-energy collision-induced fragmentation (Exploris 240, Thermo Scientific). Fragmentation data and accurate precursor mass were used to identify NEM- and IAA-labeled peptides. For data processing, SICs of all relevant NEM- and IAA-labeled peptides were generated (Byos V4.5-53, Protein Metrics Inc.). Relative site-specific quantitation of free thiols was conducted for each respective peptide by dividing the relative SIC area of the NEM-labeled peptide by the sum of SIC areas of NEM- and IAA-labeled peptides for the specific, Cys-containing peptide. Section title: 2.7.2 Imaged capillary isoelectric focusing (icIEF) Educational score: 4.1260223388671875 Domain: biomedical Document type: Study Language: en Charge heterogeneity was characterized using icIEF. The process involved mixing samples (Metalyse ® /Mingfule ® ) with carrier ampholytes, additives, and isoelectric point (pI) markers, then separating and focusing the species by a Maurice C System with a fluorocarbon-coated capillary (Bio-Techne GmbH, protein simple) upon voltage application. Reaction buffer with urea, methylcellulose, pharmalyte, phosphoric acid and pI markers was used. Samples were pre-diluted with urea and afterwards mixed with reaction buffer. Analysis was performed at fluorescence exposure time of 10 s. The icIEF system used whole-column imaging with parallel detection of absorbance and native fluorescence to quantitatively monitor the icIEF pattern of proteins, ultimately determining the charge heterogeneity of the samples, which was calculated as a percentage of three region areas. Section title: 2.7.2 Imaged capillary isoelectric focusing (icIEF) Educational score: 4.076202869415283 Domain: biomedical Document type: Study Language: en Charge heterogeneity was further characterized using icIEF after desialylation. Sialic acids were removed using the enzyme sialidase. Samples were mixed with 200 U α2-3,6,8 Neuraminidase (New England Biolabs) and incubated at 37°C before mixing with carrier ampholytes, additives, and pI markers. Then the procedure was continued as described as above. Section title: 2.7.3 Capillary gel electrophoresis under reducing (CGEr) conditions Educational score: 4.1565937995910645 Domain: biomedical Document type: Study Language: en This method separates reduced Metalyse ® /Mingfule ® species such as low-molecular-weight (LMW) and HMW species, as well as Metalyse ® /Mingfule ® main peak based on their respective molecular sizes. The LMW species are located in fragment region 1 (FR1). Samples were denatured with sodium dodecyl sulfate (SDS), reduced with DTT and labeled with a fluorescent dye by incubation. After electrokinetic injection, sample species were separated by size in a bare fused-silica capillary (SCIEX) filled with a SDS polymer that acts as a sieving matrix. Electrophoretic separation was performed by applying an electric field causing the negatively charged smaller protein–SDS complexes to migrate faster through the gel than larger protein complexes. The different sample species were detected by laser-induced fluorescence with the use of a solid-state laser (excitation wavelength: 488 nm; detection wavelength: 600 nm). The relative proportion of sample purity and product-related impurities was determined. Section title: 2.8 Statistical analysis Educational score: 3.904733419418335 Domain: biomedical Document type: Study Language: en All statistics are descriptive in nature. Our study is based on highly standardized and well-validated quality control release assays. All release assays, MS-based characterization, and Biacore LRP-1, ASGR, and MR binding were performed side by side as technical tetraplicates per lot. Biacore fibrin binding was performed side by side as technical duplicates per lot. HCP quantification by the Cygnus kits was performed side by side as a single measurement per lot. Section title: 2.8 Statistical analysis Educational score: 2.691399335861206 Domain: biomedical Document type: Study Language: en For plasmin-induced conversion and potency activation experiments, three lots of Metalyse ® and Mingfule ® were analyzed side by side. Section title: 3.1.1 Clot lysis release Educational score: 4.015554904937744 Domain: biomedical Document type: Study Language: en All three lots of Mingfule ® displayed lower clot lysis potency compared with Metalyse ® , which was used as the reference standard. On average, Mingfule ® exhibited 13.5% lower clot lysis potency (84.8%) compared with Metalyse ® (98.3%) . Section title: 3.1.2 Fibrin binding activity Educational score: 4.011997222900391 Domain: biomedical Document type: Study Language: en The individual similarity scores calculated for the three Mingfule ® lots were in the range of 37%–53% ( Table 2 ). These results demonstrate that Mingfule ® is dissimilar to Metalyse ® in fibrin binding, which may partially contribute to the difference in fibrinolytic activity compared with Metalyse ® . Section title: 3.2.1 HCP concentration determination Educational score: 4.122674942016602 Domain: biomedical Document type: Study Language: en The two HCP quantification assays demonstrated significantly higher HCP content for Mingfule ® , indicating a lower purity compared with Metalyse ® . The HCP content of Mingfule ® was up to 185-fold higher than that of Metalyse ® depending on the applied assay. In addition, initial MS-based identification of individual HCPs detected a higher number of HCPs and a higher relative abundance of HCPs for Mingfule ® ( Table 3 ). In this context, clusterin (data not shown), a protein with immunogenic potential, was detected in all Mingfule ® lots, which could not be detected in the Metalyse ® lots. Section title: 3.2.2 Monomer content quantification by HP-SEC Educational score: 4.05440616607666 Domain: biomedical Document type: Study Language: en Mingfule ® contained a lower monomer content (95.7%, SD 0.3) compared with Metalyse ® (98.2%, SD 0.1) . Mingfule ® also contained a higher content of HMW species (3.6%, SD 0.2) compared with Metalyse ® (1.0%, SD 0.1). These results further suggest that Metalyse ® has a higher purity than Mingfule ® . Section title: 3.2.3 Single-chain determination Educational score: 3.1438472270965576 Domain: biomedical Document type: Study Language: en Mingfule ® consisted of higher single-chain content (91.2%, SD 1.3) compared with Metalyse ® (73.2%, SD 3.0) . Section title: 3.3.1 Type I/II determination: Asn184 N-glycosylation occupancy Educational score: 4.017827987670898 Domain: biomedical Document type: Study Language: en The relative abundance of type I (three glycosylation sites occupied) was 40.1% (SD 0.3) and 31.5% (SD 1.4) for Metalyse ® and Mingfule ® , respectively . The relative abundance of type II (two glycosylation sites occupied) was 59.9% (SD 0.3) and 68.5% (SD 1.4), respectively. Section title: 3.3.2.1 Sialylation Educational score: 4.160322666168213 Domain: biomedical Document type: Study Language: en Compared with Metalyse ® , the sialylation for Mingfule ® was higher for the N-glycans on Asn103 (+24.5% ± 90% confidence interval 2.8%) and Asn448 (+9.0% ± 0.7%) and lower for Asn184 (−6.0% ± 1.3%) ( Table 4 ). Section title: 3.3.2.2 Galactosylation, fucosylation, and mannosylation Educational score: 4.172901630401611 Domain: biomedical Document type: Study Language: en Compared with Metalyse ® , the galactosylation for Mingfule ® was lower for the N-glycans on Asn103 (−8.1% ± 1.7%), Asn 184 (−7.5% ± 0.6%), and Asn448 (−17.0% ± 2.1%). Similarly, the fucosylation for Mingfule ® was lower for the N-glycans on Asn103 (−35.3% ± 3.0%), Asn184 (−9.7% ± 1.0%), and Asn448 (−1.1% ± 0.2%). Furthermore, for Mingfule ® , mannosylation was marginally higher for N-glycans on Asn103 (the mean offsets were +1.4% ± 0.1%) and Asn448 (+0.1% ± 0.0%) compared with Metalyse ® ( Table 4 ). Section title: 3.3.2.3 Antennary branching Educational score: 4.179655075073242 Domain: biomedical Document type: Study Language: en Compared with Metalyse ® , the bi-antennary for Mingfule ® was higher for N-glycans on Asn103 (+1.3% ± 0.7%) and Asn448 (+12.4% ± 2.6%) and lower for Asn184 (−3.1% ± 1.0%). The tri-antennary for Mingfule ® was lower for the N-glycans at all three positions (Asn 103, -2.5% ± 0.4%; Asn 184, −3.8% ± 0.5%; Asn448, −3.3% ± 0.9%). The tetra-antennary for Mingfule ® was lower for N-glycans on Asn184 and Asn448 (the mean offsets were −1.8% ± 0.2% and −8.7% ± 1.6%, respectively) compared with Metalyse ® ( Table 4 ). Section title: 3.4 Impact on clearance receptors Educational score: 4.087567329406738 Domain: biomedical Document type: Study Language: en All three Mingfule ® lots showed highly dissimilar binding to the three clearance receptors compared with Metalyse ® ( Table 2 ). Individual Mingfule ® lot similarity scores ranged from 17% to 30% for binding to LRP-1, 5% to 9% for binding to ASGR, and 8% to 10% for binding to MR. Section title: 3.5.1 Chemically induced post-translational modifications Educational score: 4.102300643920898 Domain: biomedical Document type: Study Language: en The chemically induced post-translational modifications were relatively similar between Mingfule ® and Metalyse ® , except for a pronounced difference in the abundance of free thiols ( Table 4 ). For Mingfule ® , the level of free thiols compared with Metalyse ® was −75.3% (±7.7%), indicating a substantially lower level of free thiols compared with Metalyse ® . Section title: 3.5.2 Differences in charge and size Educational score: 4.26378059387207 Domain: biomedical Document type: Study Language: en Mingfule ® displayed differences in both charge and size heterogeneity compared with Metalyse ® , displaying an average charge heterogeneity of 20.4% (SD 1.4) for region 1, 72.6% (SD 1.0) for region 2, and 6.9% (SD 0.7) for region 3. For Metalyse ® , an average charge heterogeneity of 24.1% (SD 0.9) for region 1, 64.9% (SD 0.6) for region 2, and 11.0% (SD 0.4) for region 3 was observed. After desialylation, the charge heterogeneity of Mingfule ® was shown to be 15.6% (SD 0.9), 17.7% (SD 0.7), and 66.7% (SD 1.5) for regions 1, 2, and 3, respectively. Metalyse ® displayed a different charge distribution of 17.1% (SD 0.9), 31.1% (SD 0.7), and 51.7% (SD 1.4) for regions 1, 2, and 3, respectively, and after desialylation, CGEr revealed a comparable level of LMW species (reflected by FR1) in Mingfule ® compared with Metalyse ® . As previously mentioned, HP-SEC showed that Mingfule ® displayed 95.7% (SD 0.3) monomer species, whereas Metalyse ® displayed 98.2% (SD 0.1) . Furthermore, Mingfule ® contained a higher content of HMW (aggregate) species (3.6%, SD 0.2) compared with Metalyse ® (1.0%, SD 0.1). Section title: 3.6 Plasmin-induced activation of tenecteplase Educational score: 4.153761863708496 Domain: biomedical Document type: Study Language: en In contrast to Metalyse ® , Mingfule ® is highly resistant to plasmin-catalyzed cleavage in its respective formulation buffer. After buffer exchange, the increase in clot lysis potency resulting from plasmin-catalyzed two-chain formation was approximately 30% slower for Mingfule ® in the range of linear increase. Metalyse ® achieved approximately 14% higher maximum clot lysis potency . This is in accordance with the observed difference between Metalyse ® and Mingfule ® in two-chain formation over time during incubation with plasmin. Section title: 4 Discussion Educational score: 4.155137538909912 Domain: biomedical Document type: Study Language: en Here, we compared the biochemical properties and clot lysis activity of the original tenecteplase version, marketed as Metalyse ® and TNKase ® , and a copy, Mingfule ® . Metalyse ® is approved for the thrombolytic treatment of suspected STEMI or recent left-bundle-branch block within 6 h after the onset of acute myocardial infarction symptoms, and it is also approved for the treatment of AIS within 4.5 h of the first symptoms appearing . Mingfule ® is indicated for the treatment of acute myocardial infarction, and for the fibrinolysis treatment of AIS . The current study shows that there are several significant differences between the two tenecteplase variants in terms of in vitro potency, purity, and biochemical makeup. The impact of these differences and how they may ultimately translate into clinical efficacy and safety can only be answered by direct comparison of the drugs in a clinical setting. The differences in products identified in vitro raise the question of whether data from clinical studies with one of the products can be generalized for all tenecteplase variants. Section title: 4 Discussion Educational score: 4.13528299331665 Domain: biomedical Document type: Study Language: en Comparative analyses revealed that the impurity profiles were less favorable for Mingfule ® , with more aggregates and a significantly higher HCP impurity level compared with Metalyse ® . These are key findings, as the presence of HCP impurities could potentially lead to adverse clinical effects, which may in part be attributed to HCP immunogenicity . For example, two phase III clinical trials evaluating the safety and efficacy of a recombinant biologic for the treatment of hemophilia B were suspended due to the presence of CHOP HCPs in patients treated with the drug, which clearly underscores the serious concern regulatory agencies attribute to contaminating HCPs . Section title: 4 Discussion Educational score: 4.066470146179199 Domain: biomedical Document type: Study Language: en The quantification of HCP was performed by two ELISA methods using polyclonal CHOP antibodies. It is well known that the HCP signal in ELISA shows a correlation with the amount of HCP in the sample. The ELISA signal is dependent on the specific anti-HCP antibody used in the ELISA, and therefore ELISA signals between ELISA types are variable. Although it is not possible to determine which assay provides the most accurate results without additional studies, the difference in the HCP content of Mingfule ® compared with Metalyse ® is considerable. Currently, follow-up analyses of MS-based characterization data from the HCPs detected in Metalyse ® and Mingfule ® lots are underway and may shed light on their potential impact on the quality and performance of these drugs. Section title: 4 Discussion Educational score: 4.087717056274414 Domain: biomedical Document type: Study Language: en In terms of specific impurities, clusterin, a glycoprotein associated with clearance of cellular debris and apoptosis, was detected in all Mingfule ® lots but was not detected in those of Metalyse ® . Clusterin has been shown to non-specifically interact with monoclonal antibodies (mAbs) and has been detected post-protein A purification of multiple mAb products. It is considered a high-risk HCP due to its potential to cause unwanted immune responses in patients and, in turn, may impact drug quality . Section title: 4 Discussion Educational score: 4.32053279876709 Domain: biomedical Document type: Study Language: en Analyses of post-translational modifications and sialylation patterns also differed between Mingfule ® and Metalyse ® , with potential implications for the pharmacokinetic/pharmacodynamic profile. There was a difference displayed in antennary branching, with the overall bi-antennary higher for Mingfule ® , whereas the overall tri- and tetra-antennary were higher for Metalyse ® ; differences in antennary have the potential to impact clearance of glycopeptides . There were also differences in the relative abundance of type I (three glycosylation sites occupied) and type II (two glycosylation sites occupied) molecules based on Asn184 N-glycosylation occupancy, with Metalyse ® displaying a higher type I content. Increased type I has been shown to result in reduced clearance/increased half-life of tenecteplase, and may thereby facilitate its administration as a single bolus instead of a bolus accompanied by continuous infusion . The free thiol Cys83 may influence the binding affinity of fibroblast growth factor (FGF) proteins to LRP-1 by modulating the conformation and stability beta-trefoil domain of FGF proteins . Section title: 4 Discussion Educational score: 4.246639251708984 Domain: biomedical Document type: Study Language: en Dissimilar fibrin binding affinity of Mingfule ® and Metalyse ® was observed, as well as a high degree of dissimilarity in the binding to clearance receptors LRP-1, ASGR, and MR. Differences in fibrin binding may be relevant to pharmacodynamics as well as potency, whereas affinity to the clearance receptors may potentially impact on half-life. Differences in galactosylation and sialylation can impact ASGR binding and clearance rates. ASGR and the MR (C-type 1) are well known for their selective recognition, and clearance of circulating glycoproteins and asialylated glycoproteins is selective . Sialylation of the glycoprotein, however, prevents recognition by ASGR and reduces clearance. In this context, differences in galactosylation and sialylation were noted between Mingfule ® and Metalyse ® , which are expected to translate into the observed dissimilarity in ASGR binding. In addition, mannosylation, which can impact MR binding, was higher for Mingfule ® , but the absolute values remained close to negligible. Section title: 4 Discussion Educational score: 3.677030324935913 Domain: biomedical Document type: Other Language: en Therefore, glycosylation is a critical attribute that can modulate the efficacy of a commercial therapeutic glycoprotein by balancing activity and clearance. Assuring a consistent and specific N-glycan, consisting of a spectrum of product glycans, can be crucial to achieve the desired therapeutic efficacy. Section title: 4 Discussion Educational score: 3.8203325271606445 Domain: biomedical Document type: Other Language: en For a successful thrombolytic therapy, a fine balance between activity and half-life is essential. This fine balance, resulting in a favorable safety profile, has been demonstrated for Metalyse ® since 2001, but the observed differences mean that this cannot be assumed for any tenecteplase copy without the generation of additional clinical evidence in relevant patient populations. That said, clinical studies with Mingfule ® in China have provided the basis for regulatory approval in China, with the first indication in 2015 . Section title: 4 Discussion Educational score: 4.421041488647461 Domain: biomedical Document type: Study Language: en Fundamentally, in vitro clot lysis potency was 13.5% lower for Mingfule ® (84.8%) than Metalyse ® (98.3%), potentially driven by the proportion of single-chain content of Mingfule ® (91.2%) compared with Metalyse ® (73%), as well as differences in sialic acid content and purity. Different neurologic effects of single-chain (sc-) and two-chain (tc-) t-PA have previously been discussed and are likely to have similar implications for tenecteplase. Using clinical and preclinical data, Anfray et al. demonstrated that the impact of rt-PA in stroke patients may be dependent on which form of rt-PA is administered (sc-rt-PA versus tc-rt-PA) . It should be noted that conversion of sc-t-PA to tc-t-PA does not occur at physiologically relevant rates in plasma . Thus, different levels of single-chain are expected to have different effects outside the blood clot. In this context, interaction with different receptors might be of relevance. In vitro analysis showed that the intrinsic activity of sc-t-PA selectively modulates N-methyl-D-aspartate receptor (NMDAR) signaling compared with tc-t-PA . tc-t-PA activates the MET receptor, leading to the formation of proximal complexes between MET and NMDARs, and down-regulation of glutamate ionotropic receptor NMDA-type subunit 2-NMDAR-driven signaling, whereas sc-t-PA promotes the disruption of these complexes, potentially improving neuronal survival . The physiologic effect of differences in sc-t-PA and tc-t-PA ratios is not yet fully understood. Especially for tenecteplase, available literature is limited. Section title: 4 Discussion Educational score: 4.378100395202637 Domain: biomedical Document type: Study Language: en High single-chain content may impact potency in vivo , as the full activation (or conversion to two-chain) of tenecteplase may only be achieved in a timeframe when lysis of most blood clots would have already happened. The conversion speed of sc-t-PA to tc-t-PA in the presence of purified fibrin clots is similar in scale to the duration of recanalization in AIS treatment with tenecteplase . Compared with Metalyse ® , the in vitro increase in potency by plasmin-catalyzed two-chain formation of Mingfule ® happened at an approximately 30% slower rate. This may also lead to reduced clot lysis in vivo . The root cause for the slower increase in activity is not well understood but was observed in the past for other tenecteplase products with differing quality profiles , and thus might be linked to post-translational modifications. Additionally, it is known that the content of sialic acid residues affects the biological activity of tenecteplase , and it is therefore feasible that the difference in sialylation of Mingfule ® compared with Metalyse ® may contribute to its lower clot lysis potency. Section title: 4 Discussion Educational score: 3.9775116443634033 Domain: biomedical Document type: Study Language: en Overall, the evaluation of similarity between a non-innovator biological product and its originator drug is a multifaceted process that requires pharmaceutical, non-clinical, and clinical comparative studies. The pharmaceutical comparison results should guide the design of subsequent studies, aiming to resolve any uncertainties and support the overall similarity evaluation. In this context, the tenecteplase copy, Mingfule ® , has been shown to have several meaningful differences from the originator, Metalyse ® . Therefore, with a comprehensive understanding of the clinical implications, healthcare professionals can make informed decisions regarding the generalizability of clinical results generated with different tenecteplase products. | Review | biomedical | en | 0.999995 |
PMC11695640 | Section title: 1 Introduction Educational score: 4.463083267211914 Domain: biomedical Document type: Review Language: en The eternal pursuit of finding and identifying health-promoting agents has changed how we view our food sources. We have introduced superfoods, food supplements, and nutraceuticals in developed countries, reinforcing the food industry’s further growth . Berries represent a large group of functional foods, also popularly known as “superfoods” due to their high content of disease-preventing and health-boosting chemicals . The genus Vaccinium L. (Ericaceae) includes approximately 450 diverse species, including these main commercial crops such as highbush blueberry ( V. corymbosum L.), rabbiteye blueberry ( V. virgatum Aiton, formerly known as V. ashei J.M.Reade), lowbush blueberry ( V. angustifolium Aiton), bilberry ( V. myrtillus L.), cranberry ( V. macrocarpon Aiton), and lingonberry ( V. vitis-idaea L.) . In general, berry fruit consumption has increased in recent years. Several research papers show that the increased consumption of berries is associated with a reduced risk of disorders linked with reactive oxygen species (ROS), such as cardiovascular disorders, cancer, and other inflammatory processes . A large number of scientific research cites the effect of berry consumption in three major groups: (a) physical and mental health maintenance; (b) reduction of the rate of obesity; and (c) decreased rate of chronic diet-related diseases, e.g., cardiovascular and metabolic disorders, type II diabetes . Vaccinium berries contain high concentrations of beneficial nutrients and other bioactive phytochemicals, which has led them to become the center of attraction for researchers working on the potential role of these phytochemicals in preventing chronic diseases . Many research papers have shown that these active compounds, which are present in phenolic form, are associated with high antioxidant activity. Additionally, there are several studies suggesting that wild Vaccinium Berries contain higher phenolic content and antioxidant activity than cultivated berries . Phenolic compounds present in Vaccinium berries are classified into diverse groups, which include phenolic acids such as hydroxybenzoic and hydroxycinnamic acids and their derivatives, flavonoids (flavonols, flavanols, and anthocyanins, and tannins. Tannins are further sub-grouped into condensed tannins like proanthocyanidins and hydrolysable tannins . Blueberries ( Vaccinium spp.) contain the highest amount of p -coumaric acid, chlorogenic acid, and other caffeic acid derivatives, which are types of hydroxycinnamic acids . When it comes to flavonoids among the Vaccinium berries, lingonberries ( V. vitis-idaea L.), highbush blueberries, and American cranberries ( V. macrocarpon ) are known to be the richest source of flavonols such as quercetin and myricetin derivatives and aglycones . These chemical compounds possess high antioxidant activity and play a major role in preventing many chronic diseases . However, the concentration of these compounds depends on the species, genotype, growing condition and their post-harvesting techniques . Section title: 1 Introduction Educational score: 4.280835151672363 Domain: biomedical Document type: Study Language: en Anthocyanins are important secondary plant metabolites, primarily occurring as glycosides of their aglycone anthocyanidins. The contents of the small edible berries are responsible for their bright colours as this pigment is evenly distributed in the epidermal tissues of the berries . The pigment in anthocyanins is water-soluble and responsible for orange, red, purple, and blue in fruits and vegetables . Anthocyanins are present in substantial quantities in glycosylated and various other forms in European cranberries ( V. oxycoccus ) and blueberries . European blueberries or bilberries contain fifteen anthocyanins, such as delphinidin and cyanidin monoglycosides, malvidin glycosides, petunidin, and peonidin. In the case of American cranberries, the principal anthocyanins are cyanidins, while in the case of European cranberries, it is peonidins . Similar to American cranberries, lingonberries also mainly contain cyanidin monoglycosides. Besides cyanidins, high and lowbush blueberries, lingonberries, and American cranberries contain procyanidins such as catechin and epicatechin polymers . Section title: 1 Introduction Educational score: 3.988349199295044 Domain: biomedical Document type: Review Language: en Commonly consumed Vaccinium berries have been studied for their effects on human health, but the nature and extent of their impact on humans remain vague. Hence, this article aims to provide a comprehensive overview of human clinical trials investigating the acute and chronic effects of Vaccinium berry polyphenols derived from fruits, their extracts and their derived products on inflammation, gut microbiota, diabetes, heart health, cancer, and brain activities. Section title: 2 Bioactive compounds Educational score: 4.646208763122559 Domain: biomedical Document type: Study Language: en Plants produce numerous bioactive compounds, which belong to different classes of secondary metabolites including polyphenols, phytosterols, lipoates, carotenoids, etc. . In berries, the most abundant bioactive compounds are phenolics, which are mostly found in leaves, fruits, and seeds but can also be present in other parts of plants. The chemical structure of the phenolic compounds carries one or more aromatic rings with one or more hydroxyl groups . Phenolics are either present in free or conjugated forms with water or fat-soluble compounds . Conjugated forms of phenolics are predominantly present as conjugated hydroxycinnamic acids, flavonol glycosides, and anthocyanins . These phenolics are not species-specific but shared across the genera. Among the berry polyphenols, anthocyanins constitute a large percentage. They have a characteristic C6−C3−C6 carbon structure and are glycosylated polyhydroxy and polymethoxy derivatives of flavylium salts . A study has reported that anthocyanins have a glycosidic structure containing more than two sugar molecules, such as galactose, arabinose, xylose, and glucose, that effectively connect with aglycon and form through the phenylpropanoid pathway. Anthocyanins have more than 600 compounds and more than 30 anthocyanidin compounds . The major phenolic compounds (anthocyanins) found in Vaccinium berries are listed in Table 1 . Anthocyanins are uniquely characterized by an oxonium ion on the C ring and are highly pigmented . Among these anthocyanin compounds, quercetin, myricetin and their glycosidic derivatives reach up to 30%. Anthocyanins, including procyanidins and anthocyanidins such as cyanidin, malvidin, peonidin, delphinidin, and petunidin, can account for up to 24% of all polyphenolic compounds. Phenolic acids primarily include p -coumaric acid, chlorogenic acid, caffeic acid, ferulic acid and vanillic acid. They account for up to 12% of the total polyphenols . Section title: 2 Bioactive compounds Educational score: 4.083293914794922 Domain: biomedical Document type: Review Language: en Plant phenolics were regarded as antinutritional and toxic for a long time as these compounds’ chemical nature of functioning as an inhibitor to proteolytic, lipolytic and glycolytic enzymes reduces their ability to absorb nutrients . However, the toxicity of the phenolic compounds derived from berries was generally unnoticed in the previous studies, while the benefits were observed. Apart from being a source of non-nutritive compounds such as phenolics , Vaccinium berries also contain a wide range of nutritive compounds such as simple sugars like glucose and fructose, minerals, for example, phosphorus, calcium, iron, potassium, magnesium, manganese, sodium and copper, etc. . Iron and manganese are essential components of antioxidant enzymes among these above-mentioned minerals. Moreover, berries contain vitamins A and E, reducing inflammation and acting as antioxidants . Apart from that, berries contain high concentrations of dietary fibres and low concentrations of lipids. These fibers reduce the concentration of low-density lipoproteins (LDL) in blood serum and reduce the chances of occurrence of cardiovascular and neurodegenerative diseases and cancer. All these nutritive, non-nutritive compounds, vitamins, and minerals in the Vaccinium berries synergistically affect human health . Section title: 2 Bioactive compounds Educational score: 3.9285356998443604 Domain: biomedical Document type: Study Language: en Apart from the polyphenolic compounds, Vaccinium berries also contain major lipid groups such as unsaturated fatty acids, sterols, terpenoids, and others that have high biological activity. These lipids are different from those found in mammals, which is why consuming these lipids has a significant role in human metabolism . The first report of the presence of lipids was investigated in cranberries . Klavins et al. studied the lipid profile of blueberry, bilberry, lingonberry and cranberry grown in the wild in Latvian forests and bogs. The lipid profile revealed 111 different types of lipid fractions, including fatty acids, sterols, triterpenoids, alkanes, phenolic and carboxylic acids and carotenoids. Since then, this group of compounds has been studied in many berry species. However, there is no detailed report on lipids found in Vaccinium berries. Section title: 3.1 Overview of biological activities Educational score: 4.413086891174316 Domain: biomedical Document type: Study Language: en Due to the response to the biotic and abiotic stresses, plants produce phytochemicals. They are also known as secondary metabolites. Like other fruits and vegetables, berries were found to be a great source of bioactive phytochemical components. Berry phytochemicals comprise bioactive components such as tannins, polysaccharides, alkaloids, vitamins, flavonoids, and other trace elements. They also contain sugar and fiber, which increases fruit taste and possesses many biological properties. In berries, these bioactive properties are directly related to the concentrations of the various phytochemicals in these fruits. Berry research has always been generally focused on their antioxidant properties. The antioxidant properties of these phytochemicals reduce oxidative damage to DNA, RNA, proteins and lipids at the cellular level by scavenging reactive oxygen species (ROS) . ROS are responsible for triggering aging and several inflammatory conditions, as well as cancer . They also promote the regeneration of other antioxidants and endogenous antioxidant defense systems . The imbalance between oxidants and antioxidants results in abnormalities. It produces significant ROS, including O 2 −, HO − , NO, and RO − , which interferes with the cellular processes . These superoxide ions can convert into hydrogen peroxide (H 2 O 2 ), which can further convert into the highly reactive hydroxyl radical (OH − ). Hydroxyl radicals, due to their high reactivity, cause oxidative damage, including lipid peroxidation in membranes, oxidative modification of proteins, and oxidative damage to DNA . Section title: 3.1 Overview of biological activities Educational score: 4.350792407989502 Domain: biomedical Document type: Study Language: en Various methods have been used to determine the antioxidant activity of Vaccinium berries. Among them, the Folin-Ciocalteu method, the copper ion reducibility assay (CUPRAC), the ferric ion reducibility assay (FRAP), the DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical scavenging method, and the ABTS method were mostly used in the scientific literature . Goyali et al. examined the oxidative capacity of lowbush blueberry ( V. angustifolium ). It was found that the total phenolic content (TPC) value ranged from 34.2 to 42.7 mg GAE/g FW, total flavonoid content (TFC) from 12.7 to 22.3 mg CE/g FW, and proanthocyanidin content (PAC) from 4.7 to 6.5 mg CE/g FW in the greenhouse-grown and their cutting counterparts. In another study on half-high blueberries, results of the biochemical assays of the greenhouse-grown and somatic embryogenesis-derived plants revealed that TPC varied from 0.26 to 0.46 GAE/g lw, TFC varied from 7.93 to 11.65 CE/g lw, and antioxidant activity (AA) varied from 0.08 to 14.85 GAE/g lw. The results showed that the propagation method and genotype impact the phenols and flavonoids in the leaves . A study on V. oxycoccos and V. macrocarpon compared the AA by the Folin-Ciocalteu method. It was found that polyphenol quantity in V. macrocarpon was 296.3 mg/100 g fresh weight while in V. oxycoccos , it was 288.5 mg/100 g fresh weight. However, DPPH revealed that V. oxycoccos had a stronger antioxidant potential (16.4 μmol TE/g FW) than V. macrocarpon varieties (13.08 μmol TE/g FW), which led to the inference that the concentration of resveratrol in the analyzed samples of both the species that may have an impact on the AA of the varieties . The antioxidant capacity of the Vaccinium species has been tested in in-vivo studies as well. A study was conducted on male Drosophila melanogaster to analyze the anti-aging effect of anthocyanins derived from the bilberry extracts. It was reported that the administration of anthocyanin extracts at the concentrations of 2.5, 5.0 and 10.0 mg/mL extended the life of the flies by 9.16%, 11.90% and 6.88%, respectively, compared to the control sample . Several pieces of research show that the effect of phytochemicals derived from Vaccinium berries is directly associated with their anticancerous activities . Not only anticancerous properties, but numerous studies have shown that all these components are believed to hold a broad spectrum of biomedical functions, including anti-inflammatory, antimicrobial, antiviral and antioxidant properties . Due to their health-promoting activities, these berries are highly recommended for the human diet, as their antioxidant effects have been explored in several in vitro and in vivo studies ( Table 2 ). Section title: 3.1 Overview of biological activities Educational score: 4.337346076965332 Domain: biomedical Document type: Review Language: en Studies have reported that adding fruits to our diet reduces the risk of chronic diseases like cancer, type II diabetes, obesity, and cardiovascular disorders . It was revealed that dietary intake of flavonoids is associated with a lowered risk of all-cause mortality , including CVD. However, it is important to understand that all subclasses of flavonoids are not equally involved with cardioprotection, as there is a huge gap between flavonoids’ bioavailability and bioactivity. Anthocyanins, a subclass of flavonoids, are one of the main phytochemicals present in Vaccinium berries. It was found that anthocyanins have a greater bioavailability than was previously estimated . Several in-vitro studies have shown that anthocyanins are as bioactive as their parent compounds and sometimes even more . Anthocyanins are known to stimulate anti-inflammatory, anti-atherogenic, antioxidant and vasodilatory actions . Not only that but there is also a growing body of evidence revealing that dietary inclusion of anthocyanins improves in vivo vascular health as they have a potential underlying mechanism of augmenting endothelial-derived nitric oxide (NO) bioavailability. It was found that anthocyanins can directly or indirectly increase NO availability either by upregulating endothelial nitric oxide synthase and L-arginine pathways or via optimizing nitrate-nitrite-NO pathway and reducing NO degradation by their antioxidant activities . NO is an important molecule as it regulates endothelial homeostasis. Anthocyanin displays strong antioxidant activities, and foods high in anthocyanin have been shown to improve endothelial function . Research on the effect of flavonoid intake on mortality showed that intake of anthocyanidins is positively correlated with decreased risk of CVD mortality (Summary relative risk = 0.89, 95% CI: 0.83, 0.95) when tested amongst 5 cohorts . All the available research done in vitro and in vivo on antioxidant and anticancerous properties of Vaccinium berries has advanced our understanding of their effects on human health and diseases. Therefore, the current review comprehensively summarizes what is currently known about the medicinal potential of these berries. Section title: 3.2 Anti-inflammatory effect Educational score: 4.444449424743652 Domain: biomedical Document type: Study Language: en Inflammation is known as the first line of defense in animals in response to the attack of pathogens, allergens, or any kind of tissue injury. As a result of the inflammatory response, macrophages, which are part of our immunity system, release inflammatory mediators such as interleukins, nitric oxide (NO), tumour necrosis factor-α (TNF-α), and prostaglandin (PGE2) . Usually, overexpression of such mediators is associated with a response to type II diabetes, cancer, and cardiovascular diseases (CVDs) . Over the years, much-accumulated data on pre-clinical studies of mice shows that Vaccinium berries, such as blueberries, reduce adiposity while increasing insulin sensitivity and decreasing inflammatory responses . A study on blueberries and blackberries disclosed that a daily dietary intake of 9–18.9 mg/kg BW of phenolic extract reduced cholesterol in blood plasma and metabolic dysfunctions induced by high-fat diet (HFD) in C57BL/6J mice . Another study showed that the continuous intake of Nordic wild blueberries with HFD (45% fat) for 12 weeks slows down weight gain because of obesity-induced inflammation in C57BL/6 . Similarly, HFD mixed with blueberry powder for 12 weeks helped to restore innate immune response and T-cell proliferation in HFD-induced obese mice . This also shows that consuming an adequate quantity of blueberry with an HFD may target the crucial factors in immune response and inflammation. A study in macrophages (RAW 264.7) revealed that blueberry anthocyanin reduced the expressions of two cytokines, such as Tumor necrosis factor α (TNFα) and Interleukin 1β (IL-1β), after 3 h of treatment by suppressing the NF-kB pathway expression . A study on V. floribundum revealed that phenolic extract of the berries reduced lipid accumulation and inhibited the production of anti-inflammatory response-inducing enzymes such as PGE2, NO, COX-2, and iNOS in macrophages (LPS-RAW 264.7) . The antioxidant and anti-inflammatory effects correlate with phenolic content in berries being dependent on the species, varieties, and geographical location . Section title: 3.3 Maintenance of gut microbiota and antimicrobial and antiviral activities Educational score: 4.21663761138916 Domain: biomedical Document type: Study Language: en Dietary consumption of berries is also known to help grow bacteria in the gut. Berries contain polyphenols, which promote symbiotic bacteria, reducing dysbiosis disorders . Phenolics and flavonoids present in the berries are also found to be effective against pathogenic bacteria and fungi . The human gut microbiota consists of viruses, bacteria, fungi, protozoa, and archaebacteria . A berry diet can promote the growth of beneficial microbiota and inhibit negative bacterial populations in the gut. Studies have shown that black raspberries and Vaccinium berries, such as blueberries and lingonberries, help increase the growth of Lactobacillus and some of their subspecies, the population of Bifidobacterium , and help in slowing down obesity-related problems . A study revealed that cranberry juice has a strong activity against co-adhesion and co-aggregation of oral plaque bacteria . In another study, several types of berry extracts, including lingonberry ( V. vitis-idaea ), bilberry ( V. myrtillus ), were found to have antibacterial effects against commonly found pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Listeria monocytogenes , and Bacillus cereus . Berry and leaf extracts of lingonberry have shown maximum anti-microbial effects against S. aureus and MRSA (clinical) oral cavity isolates . Research done on Romanian blueberry (var. Elliot) showed that minimum inhibitory concentration of leaf extract was discovered to be highly effective against bacterial strains such as S. aureus, Escherichia faecalis, Rhodococcus equi, E. coli , and Klebsiella pneumoniae and a few Candida fungal strains, such as C. albicans, C. zeylanoides , and C. parapsilosis . Section title: 3.4 Antidiabetic effect Educational score: 4.533349514007568 Domain: biomedical Document type: Study Language: en Diabetes mellitus (DM) is a chronic disease which is associated with other lethal diseases, including hypertension, obesity, cardiovascular diseases, and hyperlipidemia. DM is classified into type I and type II; among these, type II contributes to more than 90% of all diabetes globally . According to the World Health Organization (WHO), 108 million in 1980 and 422 million in 2014 were living with diabetes. A report done by Cho and co-workers in 2018 revealed that 451 million adults were diagnosed globally with diabetes, and it was predicted to reach up to 693 million by 2045 . Either form of DM is known to increase the risk of serious chronic illnesses such as blocking heart and blood vessels and affecting kidneys, eyes and nerve functions. This is due to the high blood sugar level, which affects and damages the nerves and the blood vessels controlling these organs. Blockage of the heart and blood vessels may also cause complications like CAD and stroke . Not only CAD but CVD is also known to be the prime cause of death in patients with DM . Several researchers have investigated and supported the idea that berry polyphenols have an antidiabetic effect, which is usually associated with glucose homeostasis. Glucose homeostasis can be regulated in an insulin-dependent and independent manner. Polyphenols derived from berries have been studied for years for their effects on insulin-dependent glucose metabolism. This can be achieved by regulating insulin secretion via modulating pancreatic-cell function and peripheral tissue sensitivity . It was found that Canadian blueberry extracts increased 3H-thymidine incorporation in TC-tet cells and increased cell proliferation by 2.8-fold . In another study, it was seen that dietary supplementation of freeze-dried whole blueberry powder in a double-blinded and placebo-controlled sensitivity had antidiabetic effects in obese, nondiabetic, and insulin-resistant human participants (p < 0.05) when administered for over 6 weeks and reported to improve insulin sensitivity . In diabetic C57b1/6J mice, feeding them a blueberry diet also displayed antidiabetic activity. Blueberry fraction enriched with phenolics and anthocyanin, in addition to Labrasol (a pharmaceutically acceptable self-micro emulsifying drug delivery system), was reported to lower raised blood glucose levels when fed to diabetic C57b1/6J mice. The hypoglycemic effect of the concoction was equivalent to that of metformin, a well-known antidiabetic drug . A study on postprandial healthy women showed that the administration of either whole lingonberries or extracts reduced sucrose-induced postprandial glucose and insulin concentrations throughout the first 30 min of consumption. Furthermore, it was seen that during the second-hour post-intake, the concentration declined slowly but improved the overall glycemic profile (p < 0.05). Additionally, the investigation showed that whole berries and extracts stopped the sucrose-induced late postprandial hypoglycemic response and the compensatory free fatty acid recovery . Schell et al. experimented with the anti-diabetic activities of cranberry extract in T2D and revealed that administration of dried cranberry significantly improved the postprandial glucose excursion. These situations revealed that the increasing incidence of DM and its associated diseases can be controlled with a Vaccinium berry-rich diet. Section title: 3.5 Cardioprotective effect Educational score: 4.505893707275391 Domain: biomedical Document type: Review Language: en Several studies have shown a strong connection between berry-derived anthocyanins and cardiovascular health. Clinical studies such as the Kuopio Ischemic Heart Disease Risk Factor Study for a follow-up of around 13 years revealed a considerably lowered risk of CVD-associated death among men who had a significantly higher quartile of berry intake (>408 g/day) than men with the lowest intake (<133 g/day) . Although these results positively impacted CVD risk factors, the models also showed an inverse correlation between the intake of fruits, berries, and vegetables and serum haptoglobin in blood, an inflammation marker . A large group of postmenopausal women (n = 34,489) contributing to a CVD mortality study associated with blueberry intake for a 16-year follow-up period at Iowa Women’s Health Study found that consumption of blueberries once a week significantly decreased coronary heart disease mortality using an age-and energy-adjusted model . The most significant conclusion drawn from all these clinical studies was that dietary inclusion of berries in everyday diet might decrease LDL oxidation and lipid peroxidation, reduce plasma glucose or total cholesterol, and increase HDL cholesterol and plasma or urinary antioxidant capacity . As a high content of plasma glucose, lipids, and lipid oxidation have been associated with coronary artery disease (CAD), these researchers suggested that edible berries, including berries from the Vaccinium family, can be consumed to reduce the risk factors of CAD significantly . It was further shown that the regular inclusion of berries in the diet also reduces the postprandial metabolic and oxidative stresses, which are also associated with CAD . Cranberry has been proven to be very effective against several health issues, including the management of systolic blood pressure in healthy men . Another study showed that cranberry extract positively affects lipid profiles in subjects with type I or II DM . Various other studies showed that consuming blueberries and cranberries significantly decreases postprandial oxidative stress, specifically lipid peroxidation . Many studies suggest the inverse correlation of flavonoid (specifically anthocyanin) intake with the occurrence of CVD and the elevated risk factors involved with CVD . Berry polyphenols positively affect the lipid profile and endothelial function of the blood vessels by reducing blood pressure and platelet accumulation . Not only that, but their antioxidant and anti-inflammatory activities also support cardiovascular health . Not only the berry extracts from fresh or frozen fruits but baked goods with lowbush blueberries ( V. angustifolium ) exhibited similar effects on endothelial function (FMV) as with the drink made with freeze-dried blueberry powder . All this available research suggests that dietary inclusion of berries can potentially be used as a therapy for pre-hypertension and hypertension management . However, none of these clinical trials were found to interfere with biomarkers responsible for inflammation, except in one case, it was found that cranberry juice supplementation substantially decreases adhesion molecules in healthy volunteers . Section title: 3.6 Anticancerous effect Educational score: 4.09234619140625 Domain: biomedical Document type: Study Language: en Among all the other healthy eating habits, including berries in the everyday diet is one of the most promising ways to prevent cancer . Phytochemicals present in the berry extracts influence genome stability at several stages, such as malignant transformation, initiation modulation, promotion and progression of cancer . In general, berry extracts combat carcinogenesis in animal models. However, when exposed to chemical carcinogens, blueberry extracts did not protect animal models. DNA damage was noticed in the tumours, and there was no evidence of a reduction in the proliferation rate of the cancerous cells or size of the tumours when pre- or co-treated with blueberry extract. These results further suggested that despite having higher antioxidant capacity than other berry species, blueberries are deficient in one or more cryoprotective phytochemicals, preventing chemically induced cancer in the animal model . Section title: 3.6 Anticancerous effect Educational score: 4.423196315765381 Domain: biomedical Document type: Review Language: en It was reported that the growth of the HT-29 cell lines in colon cancer was significantly inhibited using phenolics, such as anthocyanins and flavonols extracted from cranberry juice . In another study, lingonberry-derived quercetin and procyanidin-A2 displayed anticancerous activity against colon (HT-29), melanoma (IGR39), and renal (CaKi-1) cancer. It was also observed that quercetin demonstrated the best anticancerous activity against renal cell carcinoma (CaKi-1) . Fermented catechol extracted from fermented Rabbiteye blueberry ( V. virgatum ) extract along with Lactiplantibacillus plantarum (CK10) resulted in inducing apoptosis and inhibiting HeLa cell multiplication after 24–72 h of administration . Various flavonol compounds such as kaempferol, quercetin, and genistein acid extracted from bilberry or European blueberry ( V. myrtillus ) demonstrated cytotoxic effects against HCT-116 colon cancer cells. This study further showed that kaempferol had better anticancerous activity than other flavonols, inducing apoptosis by preventing apoptosis proteins (IAPs) inhibitors . Extracts from Vaccinium-berries such as blueberry, bilberry, cranberry, and lingonberry contain anthocyanins and ellagic acid, which are known to exhibit anticarcinogenic activities . Other reports suggest that cranberry extracts and press cake can significantly inhibit cell growth in breast, prostate, skin, brain and liver cancer cases by stopping the G1 stage of the cell cycle and initiating apoptosis . Additionally, bilberry extracts were found to induce programmed cell death in patients with leukemia . Extracts of several fruits, including blueberries, blackcurrant, black chokeberries, and raspberries, showed a strong antagonistic effect on the proliferation of breast cancer cell line MCF-7 and the colon cancer cell line HT29 and reduced their growth by up to 74% . Section title: 3.7 Neuroprotective activity Educational score: 4.314979076385498 Domain: biomedical Document type: Study Language: en There is much evidence supporting that oxidative stress of reactive oxygen species (ROS) in cells is responsible for the progression of neurodegenerative diseases such as Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease , and berry-derived antioxidants were effective against neurodegenerative diseases . Berry antioxidants also demonstrate neuroprotective activities, and several studies have shown that phenolic components from the Vaccinium species have anti-inflammatory and neuroprotective effects. In a study done with blueberry and lingonberry, brain-derived cell cultures from rats were found to be significantly tolerant against glutamate excitotoxicity when treated with blueberry extracts for 24 h. However, lingonberry ( V. vitis-idaea L.) extracts failed to provide any protection against it. Additionally, leaf extracts of blueberry and lingonberry displayed significant neuroprotective effects, while among the fruits, only blueberry fruits showed neuroprotection on the same brain cells . That is why further work has been done to investigate the neuroprotective activity of blueberry leaf extract in microglial cells derived from mice. Microglial cells are the brain’s first line of defence cells; glutamate or α-synuclein was administered to microglial cells to induce an inflammatory response. The cells were treated with blueberry fruit and leaf extracts, which decreased cell death and reduced inflammation after 24 h. This result further points to the fact that a blueberry-rich diet, with leaves or fruits, can protect against neurodegenerative disorders . Over the years, studies have suggested that the inclusion of blueberries in a regular diet may help with age-related and oxidative stress, which are responsible for declined brain function . Another report suggested that a blueberry-supplemented diet can improve behavioural deficits associated with age or a high-fat diet . Overproducing ROS and reactive nitrogen species (RNS) free radicals cause aging and neurodegenerative diseases. Cortical cell cultures derived from neonatal rat pups were intoxicated with glutamate for 24 h, and it was seen that glutamate was responsible for morphological disruptions such as increased formation of dark punctae and disruption of cell bodies. Glutamate-treated cells were administered with lingonberry and blueberry leaf and fruit extract. They were found that, while lingonberry fruits failed to provide any protection from glutamate toxicity, the leaf extracts from both the berries and blueberry fruit extract displayed no cell death in the presence of glutamate . Section title: 3.7 Neuroprotective activity Educational score: 4.1693339347839355 Domain: biomedical Document type: Study Language: en It was found that aged rats fed with blueberries have shown a reduction of ischemia-induced apoptosis in brain cells which is due to their capability of interacting with ROS and RNS, which accumulated during the ischemic phase in the central nervous system . Krikorian et al. reported in older humans that improved memory capabilities were detected by increased synaptic plasticity as a result of microglial modulation of the microglia-neuron crosstalk through the increase of the expression of CX3CR1 receptor was associated with a blueberry rich diet . Memory loss is often associated with oxidative damage to lipids, proteins, and nucleic acids. Oxidative damage to all three can disrupt neural function. It was seen that bilberry extract was significantly effective against oxidative damage by decreasing lipid peroxides and increasing superoxide dismutase activity in the brain. Additionally, it was found that long-term supplementation of bilberry extract in the diet of the OXYS rats prevented learning and memory deficits . These findings specifically signify the effect of Vaccinium berry antioxidants on the neuroprotection of brain function. Section title: 4 Conclusion Educational score: 4.162168502807617 Domain: biomedical Document type: Review Language: en Several in vitro and in vivo studies now indicate that berries positively impact human health by acting as strong anticancer and antioxidant agents. They are an ideal dietary source of bioactive components and could play a role in reducing cancer risk. The unique phytochemical constituents in berries act individually or synergistically to provide protection against several health disorders, including cancer and CADs. It is evident from this review that a lot has been done in this direction, but much more needs to be done to pinpoint the molecular mechanisms associated with the most beneficial phytochemicals that make up these nutritious fruits. The review summarizes the effects of bioactive compounds present in Vaccinium berries and their function against cardiovascular and neurodegenerative diseases. It was seen that measurable criteria like total anthocyanin or total phenolic content and total antioxidant content may also be associated with the effectiveness of health benefits. Overall, more in vivo data are required to understand the mechanisms of action, while more human clinical trials using different parameters such as gender, age, and any pre-existing condition should be performed such new information on the bioactive components of berries can be revealed, and the existing information could be validated. Also, using berry phenolic compounds as antimicrobial agents provides many possibilities for use in the food and medical industry. It will also be a very interesting topic for future research priority by developing new ways for berry compounds to avoid and manage antibiotic-resistant infections. In addition to the phenolic compounds, phytosterols are well-known for their antioxidant activities. Epidemiological and experimental reports suggest that they help reduce cholesterol and potentially protect against several types of cancer. Furthermore, berry lipids are also used in many commercial products. These ignited a general interest in studying these compounds in depth to understand their potential application in cosmetics, pharmacy and the food industry. | Review | biomedical | en | 0.999998 |
PMC11695645 | Section title: Introduction Educational score: 3.980339765548706 Domain: biomedical Document type: Review Language: en Obesity, a chronic, relapsing disease, is a major health issue that has increasingly become more prevalent in recent decades . The disease has several possible causes, including but not limited to genetic, metabolic, and environmental factors . The condition is marked by an abnormal accumulation of fat leading to an excessive increase in a person’s weight, attaining a body mass index (BMI) of 30 kg/m 2 or more . According to the World Health Organization, about 890 million people were obese globally in 2022, with 16% of adults aged 18 and above living with obesity . The prevalence of obesity has been particularly increasing in the developed world, with about 41.9% of adults in the United States being affected by obesity, while about 10.3% of adults aged 20 and above are affected by severe obesity . In Europe, about 60% of adults are either overweight or obese . Section title: Introduction Educational score: 3.66536283493042 Domain: biomedical Document type: Review Language: en In Saudi Arabia, obesity has also become a huge challenge, with about 23.7% of people aged 15 and above being diagnosed with obesity . According to the WHO, Saudi Arabia has the second highest prevalence of obesity in the Gulf region. Poor dietary habits and technology-inspired sedentary lifestyles are the main reasons for the recent upsurge in obesity . Given the level of these lifestyle changes, coupled with other factors such as genetic and environmental factors, the challenge of obesity may continue to be even more of a public health concern in the future. Obesity comes with a significant burden because not only does it lead to increased medical costs but also causes decreased productivity among patients . In addition, obesity is associated with other healthcare challenges, such as diabetes, non-alcoholic fatty liver disease (NAFLD), cancer, chronic kidney disease, and many others . Obesity and all the other related conditions significantly reduce patients’ quality of life and life expectancy, given that obesity is linked to many premature deaths globally. Section title: Introduction Educational score: 2.4127566814422607 Domain: biomedical Document type: Study Language: en Given the great public health threat posed by obesity, it is important that all people have adequate awareness of the condition and have sufficient information regarding avoiding it and managing weight. Saudi Arabia, being one of the most developed countries in the Gulf region, has been proven to have a major overweight and obesity challenge that poses a significant threat to the future sustainability of the country. In this regard, this study seeks to explore the awareness of obesity and weight loss management among adults in the Asir region of Saudi Arabia. Section title: Materials and methods Educational score: 1.3672783374786377 Domain: biomedical Document type: Other Language: da Study design Section title: Materials and methods Educational score: 2.3955371379852295 Domain: biomedical Document type: Study Language: en A community-based cross-sectional study was designed to assess awareness and perceptions toward obesity and weight loss management among adults in the Asir region, Saudi Arabia. The research was conducted over seven months between the period of April 2024 to October 2024. Section title: Materials and methods Educational score: 2.023653507232666 Domain: biomedical Document type: Study Language: en Inclusion and exclusion criteria Section title: Materials and methods Educational score: 2.225646734237671 Domain: biomedical Document type: Study Language: en The study targeted adult residents of the Asir region, Saudi Arabia, aged 18 and above, inclusive of both genders. Inclusion criteria required participants to be willing to participate and capable of completing an online survey independently. Exclusion criteria included individuals under the age of 18, pregnant women, individuals who participated in the pilot study, and those who declined participation. Section title: Materials and methods Educational score: 1.664145588874817 Domain: biomedical Document type: Study Language: fr Sample size and sampling technique Section title: Materials and methods Educational score: 2.9878697395324707 Domain: biomedical Document type: Study Language: en The study employed a non-probability convenience sampling method in recruiting participants. The sample size was determined using the Cochrane sample size formulae with a 95% confidence level and a 5% margin of error, resulting in a minimum acceptable sample size of 384. However, to improve the reliability, a higher sample of 638 was used. Section title: Materials and methods Educational score: 1.335404396057129 Domain: biomedical Document type: Other Language: en Data collection tools and methods Section title: Materials and methods Educational score: 3.2676405906677246 Domain: biomedical Document type: Study Language: en Data were collected using a self-administered electronic questionnaire distributed via Google Forms (Google LLC, Mountain View, California, United States). The questionnaire, modified to suit the study objectives, was reviewed by two research faculty experts from the University of Bisha. A pilot study involving 30 participants was conducted to ensure the questionnaire’s clarity and relevance. These participants were excluded from the main study. The pilot yielded a Cronbach's alpha of 0.841, indicating high internal consistency. The questionnaire was distributed via WhatsApp groups and promoted by local social influencers to encourage participation. The survey collected sociodemographic information and assessed participants’ awareness regarding obesity, weight loss management, and history of weight loss surgery. All questions and consent forms were translated into Arabic to facilitate understanding among participants and also back translation was done to English. Section title: Materials and methods Educational score: 0.9184456467628479 Domain: biomedical Document type: Other Language: tl Data analysis Section title: Materials and methods Educational score: 2.347849130630493 Domain: biomedical Document type: Study Language: en Microsoft Excel spreadsheets (Microsoft Corporation, USA) were used to do all the data cleaning. Then, data were transferred to IBM SPSS Statistics for Windows, Version 27.0 where the analysis was conducted. As the data were categorical, it was presented in tabular form, displaying counts and frequencies. Appropriate statistical tests like the chi-square test were applied to determine statistical significance set at p < 0.05. Section title: Materials and methods Educational score: 1.1492395401000977 Domain: other Document type: Other Language: en Ethical considerations Section title: Materials and methods Educational score: 1.4611154794692993 Domain: biomedical Document type: Other Language: en Ethical approval for the study was obtained from the University of Bisha Institutional Review Board (IRB) (approval no. UB-RELOC H-06-BH-087/). No study activities commenced before securing IRB approval. All participants were informed about the study's objectives, and individual informed consent was obtained prior to survey completion. Participants were assured of their right to withdraw from the study at any point, with confidentiality and anonymity maintained throughout. Respondents’ data were protected, and their right to refuse or withdraw from the study was respected. Section title: Results Educational score: 3.0112855434417725 Domain: biomedical Document type: Study Language: en Table 1 provides an overview of the sociodemographic and self-reported characteristics of the 638 participants in this study. Females represented the majority of the sample at 69.6%, with males comprising 30.4%. The largest age group was 18-20 years (38.6%). In terms of the BMI, 47.8% of the participants had a normal BMI (18.5-25), while a smaller proportion, 2.2%, had a BMI over 40. Regarding weight perception, 48.8% of the participants considered their weight to be normal, 39% felt they were overweight, and 5.8% reported feeling very overweight. Section title: Results Educational score: 3.8974525928497314 Domain: biomedical Document type: Study Language: en Table 2 shows that an overwhelming majority (83.5%) reported not following any specific diet, while only 16.5% adhered to a dietary regimen. Regular physical activity was also low, with just 29.8% engaging in regular exercise. Among those who exercised, only 4.7% exercised daily with the majority nearly half of them (47%) not exercising at all. Sleep patterns varied, with slightly more than of the respondents (51.9%) sleeping between three and six hours per day and 46.1% achieving seven hours or more. Regarding hydration, 61.9% of the participants did not meet the recommended daily water intake, with only 38.1% meeting or exceeding the recommended levels. Finally, in terms of daily activity levels, 44.8% described their lifestyle as lightly active, followed by 30.3% who were moderately active, while a smaller group was sedentary (19.4%) or very active (5.5%). Section title: Results Educational score: 3.6888628005981445 Domain: biomedical Document type: Study Language: en Table 3 depicts a notable lack of knowledge among the respondents regarding certain medications, such as antidepressants, nearly half (49.1%) believe that can lead to obesity, while 8.6% disagreed, and 42.3% were unsure. A majority (66.1%) agreed that medical conditions like metabolic syndrome, polycystic ovary syndrome, and hypothyroidism can result in weight gain, with only 4.4% disagreeing and 29.5% expressing uncertainty. Regarding the impact of stress, 45% of the participants felt that high stress levels could contribute to obesity, while 21% disagreed and 34% were unsure. Similarly, 46.2% believed that inadequate quality sleep could lead to obesity, whereas 18.8% disagreed, and 35% were uncertain. Section title: Results Educational score: 2.8181099891662598 Domain: biomedical Document type: Study Language: en Table 4 shows that a small fraction of participants followed popular diets, with 1.6% following the Dukan diet and 1.4% following the Atkins diet. A larger group, 24.5%, practiced intermittent fasting, while 8.2% reported following diets other than those specifically listed. Similarly, 8.2% of the participants used herbal medicines as part of their weight loss efforts. By contrast, weight loss surgery was rare, with only 1.7% of the participants having undergone such a procedure. Section title: Results Educational score: 3.9651331901550293 Domain: biomedical Document type: Study Language: en As per Table 5 , the most common choices were avoidance diets (32.7%) and calorie-focused diets (21.2%), with smaller portions opting for low carbohydrate diets (19.2%) and medically recommended diets (7.7%). Regarding herbal and natural remedies for weight loss, the most frequently used were herbal teas and natural supplements like green tea and apple cider vinegar (32.7%), while others combined herbs such as ginseng, psyllium, and ginger (25%). The majority of participants (92.9%) reported not using any medication for weight loss. Among those who used medications, the most commonly used were Saxenda (1.9%), Ozempic (1.7%), Monjaro (1.6%), and other unspecified medications (1.6%). Victoza was the least used medication, with only (0.3%) of participants reporting its use. Section title: Results Educational score: 4.060944557189941 Domain: biomedical Document type: Study Language: en Table 6 shows that gender differences were evident, with females showing higher levels of good knowledge (73.5%) compared to males (26.5%), with a significant p-value of 0.003. Age was also significantly associated with knowledge levels (p < 0.001), where participants aged 21-30 had the highest proportion of good knowledge (43.9%), while younger participants (18-20) were more likely to have poor knowledge (50.4%). Weight showed a significant relationship (p = 0.011), with individuals in the 50-69 kg range having a higher prevalence of good knowledge (46.4%). BMI also correlated with knowledge levels (p = 0.008); participants with a normal BMI (18.5-25) had higher knowledge levels (48.3%), while those uncertain of their BMI or in higher ranges showed lower knowledge. Self-perceptions of weight were similarly associated, as those identifying as overweight were more likely to report good knowledge (44.7%, p < 0.001) compared to those who perceived their weight as normal (43.4%). No significant associations were found for height, indicating it had minimal impact on knowledge levels. Section title: Discussion Educational score: 4.052084922790527 Domain: biomedical Document type: Study Language: en It is imperative to understand the public awareness of obesity and weight management to develop effective health interventions, especially in regions where obesity rates are high . This study focused on the awareness levels among adults in the Asir region, Saudi Arabia, to assess their knowledge and perceptions regarding obesity and weight loss management. This study found that 64.6% of the respondents have a good level of knowledge regarding obesity and weight loss. Similarly, recent research among adults in the western region of Saudi Arabia reported a comparable level of understanding, with 71.8% of participants demonstrating good knowledge . In our study, healthcare professionals and medical students scored notably higher than average, indicating stronger awareness in these groups. By contrast, a study by Duante et al. in the Philippines found that 31.7% of participants had poor knowledge of obesity and weight management, reflecting a lower level of understanding compared to our findings . It is worth noting that differences in scoring criteria, sample populations, and methodologies may account for the variation in results across studies. Section title: Discussion Educational score: 3.9705650806427 Domain: biomedical Document type: Study Language: en Our study revealed low adherence to consistent physical activity routines, a large majority of participants (83.5%) reported not following any specific diet, and only 29.8% engaged in regular physical activity. Of those who exercised, nearly half (47%) reported no exercise days per week, and only 4.7% exercised daily. While the awareness of obesity and weight management is relatively high, practical adherence to healthy habits remains low, pointing to potential gaps between knowledge and behavior. A similar study conducted among Saudi residents revealed a comparable trend, showing that while individuals demonstrated a good understanding of the importance of physical activity in managing obesity, only a small percentage (38.65%) were actively committed to weight loss efforts . Section title: Discussion Educational score: 3.9081974029541016 Domain: biomedical Document type: Study Language: en Our study further revealed that 43.4% of respondents recognize the relationship between obesity and diet, while 45% believe that high levels of stress can contribute to obesity, identifying stress as a significant risk factor. These findings indicate that although there is some awareness of lifestyle-related causes of obesity, gaps remain in understanding the full range of contributing factors. A study by Acosta et al. supports this observation, reporting that 49.1% of respondents identified age, high stress levels, and consumption of greasy foods as key risk factors for obesity . Section title: Discussion Educational score: 4.038761138916016 Domain: biomedical Document type: Study Language: en This study found that a significant proportion of respondents (92.9%) believe that anti-obesity medications, such as Saxenda and Victoza, are unsafe and less effective for managing obesity compared to following a healthy diet and exercising. In addition, most respondents did not prefer using medications for weight loss. By contrast, 1.7% of the respondents held positive views toward weight loss surgery, having previously undergone the procedure. These findings align with prior research by Lean et al., which reported that the majority of the participants (88.6%) preferred weight loss through exercise and diet over surgical methods. While Lean et al. suggested that certain weight loss medications could be viable options, they also acknowledged safety concerns . Moreover, the potential for adverse effects from long-term medication use is notable. For instance, the U.S. FDA ultimately banned the weight management drug lorcaserin due to its association with an increased cancer risk . Section title: Discussion Educational score: 4.0865068435668945 Domain: biomedical Document type: Study Language: en The results indicate a statistically significant relationship between age, self-perception of weight, and respondents' knowledge of obesity and weight loss management (p < 0.001). Specifically, younger participants in the 21-30 and 18-20 age groups demonstrated higher levels of knowledge (43.9% and 32%, respectively) compared to other age groups probably because the younger generation has increased computer literacy and engagement with technology, which potentially provides greater access to information on health topics. Gender also displayed a marginally significant association with knowledge levels (p < 0.05), with female participants achieving a higher knowledge score (73.5%) than males (26.5%). BMI was similarly associated with knowledge levels with the normal range (18.5-25) exhibiting greater knowledge levels (48.3%, p = 0.008). In addition, individuals who perceived themselves as overweight were more likely to report good knowledge (44.7%, p < 0.001) compared to those who considered their weight normal (43.4%). By contrast, height was not significantly associated with knowledge about obesity and weight loss management (p > 0.05), aligning with previous research findings that also found no correlation between height and knowledge in medical contexts . Section title: Discussion Educational score: 2.3520238399505615 Domain: biomedical Document type: Study Language: en The study has some considerable limitations. Since it was an online cross-sectional study, it could not determine cause-and-effect relationships between variables, and at the same time, it could not change variables over time. In addition, the online format may introduce selection bias, as individuals without Internet access or those less inclined to engage online were likely excluded, potentially limiting the generalizability of the results. Section title: Conclusions Educational score: 4.042810440063477 Domain: biomedical Document type: Study Language: en The study revealed a moderate level of awareness of obesity and weight management among adults in the Asir region. Despite reasonable awareness, practical adherence to healthy behaviors, such as regular physical activity and dietary management, remains low, indicating a gap between knowledge and lifestyle practices. Most participants recognize lifestyle-related factors contributing to obesity and favor non-surgical, non-medication-based weight management. The study found that variables such as body mass index, gender, age, self-perception of weight, and individual attitudes toward personal weight are associated with a greater understanding of obesity and weight management practices. These factors could therefore be strategically leveraged to target populations with lower awareness and to reduce societal stigma around obesity. | Review | biomedical | en | 0.999997 |
PMC11695668 | Section title: INTRODUCTION Educational score: 3.794616937637329 Domain: biomedical Document type: Review Language: en Electroconvulsive therapy (ECT), the oldest somatic intervention in psychiatry, continues to remain a highly effective treatment for mood and psychotic disorders. 1 , 2 Practitioners frequently encounter medical conditions that require precaution during ECT. Published reports have, however, formed a piecemeal literature with reports scattered across various physiological systems and individual disease conditions. This account collates the results into a single comprehensive review. Although the contraindications for ECT form well‐trodden ground, findings accrued since past reviews make it an evolving landscape. Section title: INTRODUCTION Educational score: 4.341335296630859 Domain: biomedical Document type: Review Language: en ECT is an enigmatic treatment in medicine: it has an impressive safety record despite profound neurochemical and hemodynamic changes associated with seizures, such as a 15‐fold rise in adrenalin and up to two times increased blood pressure (BP). 3 , 4 , 5 In contrast to the public fear of death from ECT, which was present in 20% of participants in one study, and a lay viewpoint that electric current is passed on to the brain, mortality directly related to ECT is extremely rare. 6 , 7 , 8 The skull, with a relatively high impedance, attenuates the electric flow to the brain during ECT. Most of the current is short‐circuited around the skull. 9 Therefore, the voltage gradient across a single neuron cell body drops to 0.006 V, far below the action potential required for depolarization. 10 It is rapid kindling of neurons induced by repeated pulses of current that eventually results in seizure. 11 Since the properties of electric current entering the brain are within physiological parameters, and the temperature rise in the intracranial tissues is negligible, medical complications of ECT attributed to the current per se are very few, such as electric current induced bradycardia. They mainly arise from three factors: convulsions, hemodynamic alterations, and effects of anesthesia. 6 Even when adverse effects occur, attribution to ECT is confounded by other medical factors. 12 Section title: MATERIALS AND METHODS Educational score: 3.538163900375366 Domain: biomedical Document type: Study Language: en We conducted the literature search using the following keywords: “Electroconvulsive therapy OR ECT,” “contraindications,” and “medical precautions.” Additionally, we performed searches for individual systems and conditions, such as “cardiovascular,” “aneurysms,” “arrhythmias,” “myocardial infarction,” “cardiac failure,” “aortic stenosis,” “deep vein thrombosis,” “DVT,” “aortic aneurysm,” “stroke,” “intracranial space‐occupying lesions,” “delirium,” “dementia,” “skull defects,” “myasthenia gravis,” “myopathies,” “fractures,” “pacemakers,” “implant,” “pregnancy,” “epilepsy,” “medications,” “kidney diseases,” “pheochromocytoma,” “intraocular pressure,” “obesity,” “infection,” “COPD,” and “COVID 19.” The search was limited to English‐language and human studies. Section title: MATERIALS AND METHODS Educational score: 4.019473075866699 Domain: biomedical Document type: Review Language: en To reduce the risk of selection bias, we reviewed previous reviews and practice guidelines on ECT administration in the presence of medical conditions. 13 , 14 , 15 , 16 , 17 Using the OVID search tool, we searched for each condition in PubMed and EMBASE till March 2024. All abstracts were screened and, when relevant, two independent reviewers evaluated the full texts. Discussions with a third reviewer resolved disparities. Due to significant heterogenicity of the topic, we adopted the state‐of‐the‐art (SOTA) review. A SOTA review is a type of literature review that emphasizes the most recent and innovative developments in a specific field or research area, such as “the application of ECT in complex medical conditions.” Although we conducted a comprehensive and systematic search, our primary focus remained on recent advancements, emerging trends, and cutting‐edge findings, ensuring alignment with the principles and purpose of a SOTA review. This approach allowed us to capture the forefront of current knowledge and highlight key innovations within the field. Section title: MATERIALS AND METHODS Educational score: 2.7644221782684326 Domain: biomedical Document type: Review Language: en Using the COVIDENCE tool, we initially identified 1803 studies. A large proportion of these were excluded in the initial screening due to being opinion pieces, commentaries, unstructured reviews, conference presentations, or unstructured case studies. After this preliminary filtration, 376 studies remained for full‐text review. Ultimately, only 124 papers were found to be relevant for this review . Section title: Cardiovascular diseases Educational score: 4.41867733001709 Domain: biomedical Document type: Review Language: en ECT was given in almost all cardiac conditions. 16 Most of the cardiovascular complications associated with ECT are minor and manageable. 17 The electrical stimulus is parasympathetic, causing transient asystole and bradycardia lasting for several seconds, followed by sympathetic activity resulting from seizure. 18 The catecholamine surge may persist for minutes, translating into various hemodynamic changes, namely, tachycardia, hypertension, and increased cardiac contractility. 19 BP may rise twofold, and heart rate by 1.45 times. 5 Enhanced cardiac contractility may increase mean cardiac output up to 81%. 4 All these changes can be effectively blocked by β‐adrenoceptor antagonists. 5 With bifrontal stimulation, sympathetic stimulation occurs without a preceding phase of significant bradycardia (Table 1 ). 18 , 20 (a) Arrhythmias The reported incidence varied from 8% in healthy people to 80% in patients with pre‐existing cardiac diseases. 21 , 22 The most common complication is ventricular ectopic. 22 More serious arrhythmias reported from one study of 2279 patients who were given 17,394 ECTs included second degree AV block ( n = 2), bradycardia ( n = 2), ventricular tachycardia ( n = 2), ventricular fibrillation ( n = 1), prolonged asystole, and cardiac arrest ( n = 1). 12 Notably, no one died during or soon after ECT. Another review of 80 patients showed ECT was relatively safe in older patients with pre‐existing cardiac risk factors. 36 Persistent ECG changes or an elevation of cardiac enzymes were not observed after ECT. 37 , 38 (b) Myocardial infarction Fatality in the minutes after ECT was documented in relatively old literature in a person who had an autopsy‐proven myocardial infarction. 23 Left ventricular hypertrophy was present in this case. There is a risk of cardiac arrhythmias, recurrent infarction, and myocardial rupture with ECT, particularly in the first 10 days after infarction. This risk is deemed to be reduced after 3 months. 15 However, ECT was safely given without dose titration for a patient with catatonia 4 weeks after myocardial infarction using a β‐adrenergic antagonist and close monitoring of cardiac parameters. 24 (c) Cardiac failure In a case series, ECT was not associated with any sentinel event in five patients with congestive cardiac failure. 15 Published data support the safety of ECT with cardiac ejection fraction as low as 15%, including in older individuals. 25 In such instances, ECT was administered with medications that reduce cardiac workload (e.g., lisinopril) or β‐blockers that decrease tachycardia and hypertension. (d) Aortic stenosis A case series of 10 patients and a single case report demonstrated the safe administration of ECT in severe aortic stenosis. 39 , 40 Two patients had low BP 1 min after ECT, but this was successfully managed. (e) Deep vein thrombosis ECT is an effective treatment for mood disorders and catatonia. These conditions can be associated with diminished mobility and deep vein thrombosis (DVT). 41 Pulmonary embolism (PE) due to dislodgement of the thrombus during convulsion is a known theoretical risk in the presence of DVT. In line with this, there are documented instances of PE developing after ECT, including fatal ones. 26 , 27 , 28 , 29 However, in several patients with DVT, ECT was safely administered. 41 , 42 The outcomes of ECT in the presence of DVT are thus mixed and form an area that warrants further investigations, given the lethal nature of PE. A case report showed sudden death after ECT, which was possibly linked to DVT in the context of COVID‐19 infection, but without a definite conclusion. 43 Possible contributing factors to embolization in DVT include the above‐knee location and size of the thrombus. The commencement of anticoagulants does not offer absolute protection from PE due to an already‐developed venous thrombus, which may still dislodge during convulsive movements. If the clinical indication for ECT is compelling, discussion with vascular surgeons and anesthetists is imperative to achieve complete muscle relaxation and optimal outcomes. (f) Aortic aneurysm A case series of eight patients suggested that ECT was safe in people with aortic aneurysms with aortic diameters ranging from 3.3 to 5.3 cm. 44 Section title: Risk mitigation in the presence of cardiovascular diseases Educational score: 4.02485466003418 Domain: biomedical Document type: Other Language: en A waiting period of 3 months after myocardial infarction is regarded as safe for ECT. 15 Cardiovascular complications can be mitigated by avoiding multiple stimulations and using β‐blockers to attenuate hypersympathetic response in high‐risk populations. Patients should be advised to take these medications the morning before treatment with sips of water. 16 Dose titration may be avoided, or bifrontal placement may be considered for patients at risk of asystole, such as those taking β‐blockers. 25 After successfully treating patients with cardiac failure with an ejection fraction varying from 20% to 25%, Stern and colleagues developed a protocol that included administering regular cardiac medications 60–90 min before ECT and avoiding anticholinergic drugs. 45 Section title: Cardiac monitoring during ECT Educational score: 4.056316375732422 Domain: biomedical Document type: Study Language: en Given the significant hemodynamic alterations, in particular seizure‐related catecholamine surge, cardiac monitoring is an essential part of ECT. The monitoring normally entails electrocardiography, pulse oximetry, and noninvasive intermittent BP measurements. 46 This level of monitoring does not capture rapid hemodynamic changes. Novel noninvasive devices, such as Nexfin HD, can continually monitor cardiac output and beat‐to‐beat arterial BP. Since the hemodynamic changes associated with ECT are short‐lived, techniques that use continuous monitoring to detect rapid changes can be useful, especially in patients with high‐risk conditions. Patients with low cardiac ejection fraction will require monitoring of BP concurrently with both an arterial line and dual BP cuff monitoring using monitors on both arms with continuous BP cycling. 47 Esmolol, a β‐adrenergic blocker, attenuated an increase in heart rate and systolic, diastolic, and mean arterial BP from a dosage of 1.0 mg kg −1 onward. 48 Section title: Neurological and neurosurgical conditions Educational score: 4.453344345092773 Domain: biomedical Document type: Review Language: en (a) Aneurysm and stroke So far, 27 cases of ECT in patients with aneurysms have been published. 30 ECT was safe with secured and unsecured aneurysms up to a size of 2.1 cm, except in one instance where an 84‐year‐old patient died from subarachnoid hemorrhage following rupture of an anterior cerebral artery aneurysm of 1.9 cm 2 days after 10th right unilateral ECT. 31 , 32 The patient received esmolol; the peri‐ictal BP increased to 207/89 mm Hg. This report suggests that ECT is not universally safe in patients with aneurysms. The risk appears to increase with large aneurysms. One case report demonstrated the safety of ECT within 2 weeks after stroke. 49 Another comparative study showed no difference in the delirium incidence between ECT given after stroke and without stroke; all cases of post‐ECT delirium during the poststroke period were seen after caudate stroke. 50 (b) Intracranial space‐occupying lesions An earlier review of reports published until 1984 showed complications in 74% ( n = 38) and mortality in 8% ( n = 29) of patients with intracranial neoplasms after ECT. 33 Complications included aphasia, ataxia, hemiplegia, delirium, and coma. Although the authors suggested that ECT could have precipitated deaths in 8%, some of them had aggressive tumors like glioblastoma. A more modern systematic review and retrospective data found that ECT was largely safe, with no mortality in patients with intracranial tumors. 34 , 35 Buday et al. reviewed 40 reports published until 2019. Reversible adverse reactions reported in six patients (15%) were secondary myoclonic seizure, ping‐pong gaze, Todd's paralysis, delirium, and coma. Differences between the earlier review and Buday et al. were prior knowledge of tumors, more benign tumors, dose titration, and use of dexamethasone in the latter. 33 , 34 The largest reported tumor was 8.1 × 7.6 × 4.1 cm, and ECT was not associated with adverse effects in this case. 51 A mild mass effect does not appear to preclude ECT if there is no midline shift. 52 Case reports suggest that ECT is safe in idiopathic intracranial tension. 53 (c) Delirium and dementia Although ECT may cause transient postictal confusion, there is no evidence to suggest that delirium and dementia are contraindications for ECT. The evidence indicates that ECT is beneficial in reducing behavioral symptoms in a subgroup of patients with these conditions. 54 , 55 (d) Skull pathology Skull defects can cause the direct entry of electricity into the brain with severe consequences. Given its high impedance, the skull attenuates the current flow so that only a small portion enters the brain. 9 Patients may be regularly screened for skull deficits because of trauma or surgery and lytic skull lesions. Section title: Risk mitigation Educational score: 4.102555274963379 Domain: biomedical Document type: Study Language: en A retrospective study identified one abnormal scan that prevented ECT out of 108 scans (0.93%) performed as routine pre‐ECT imaging. 56 Considering such a low probability and the relative safety of ECT, brain imaging may be performed for patients at risk, for instance those with prior aneurysms, heritable disorders associated with intracranial aneurysms, neurological symptoms such as headaches, and those above 60 years of age with a history of hypertension or smoking. Neurology and cardiology consultations are required in such situations. While esmolol is administered, intra‐arterial BP monitoring at the first session of ECT to understand the exact real‐time variation of BP during the preictal, ictal, and postictal phases is required to determine sufficient esmolol dose for subsequent sessions. Risk management in the presence of intracranial tumors includes the administration of dexamethasone to reduce edema around the tumor and phenytoin to reduce the risk of recurrent seizures. 34 An interdisciplinary approach encompassing consultations with neurologists, neurosurgeons, and anesthetists may reduce the risk of complications (Table 1 ). Section title: Neuromuscular disorders Educational score: 4.513269424438477 Domain: biomedical Document type: Review Language: en 1. Myasthenia gravis Myasthenia gravis (MG) is an autoimmune disorder characterized by antibodies against nicotinic acetylcholine receptors (anti‐AChAb), affecting the neuromuscular junction. Case reports suggest ECT is safe in patients with MG, provided adequate precautions are taken. 57 , 58 During the active phase of the disease, ECT should be avoided, and the patient should be in reasonable remission before undergoing ECT. The patient's condition can be managed through expert opinion from a neurologist and regular monitoring of pulmonary function tests, plasma pseudocholinesterase levels, and anti‐AChAb levels. 58 Anticholinergic agents are usually avoided to prevent a cholinergic crisis. Although suxamethonium was safe during remission, response to succinylcholine during ECT was variable, and in one instance, rocuronium was used with sugammadex reversal. 57 , 59 A significant reduction in endplate receptors at the neuromuscular junction may limit the action of succinylcholine, causing resistance or decreasing the safety margin. 60 Prednisolone and cholinesterase inhibitors, commonly used in MG, can prolong the half‐life of succinylcholine and result in longer‐than‐usual neuromuscular blockade. 60 , 61 Cholinesterase inhibitors can increase the risk of asystole and bradycardia, and in these situations, bifrontal ECT and stimulation without titration may be considered. 62 2. Inflammatory myopathies In patients with inflammatory myopathy, neuromuscular blockers require caution due to potential complications such as delayed or prolonged effects, hyperkalemia, diaphragmatic dysfunction, impaired coughing, and swallowing difficulties requiring special anesthetic care. These concerns arise from a rise in serum creatine phosphokinase (CK) after ECT. 37 , 63 Empirical evidence also suggests that CK levels normalized with subsequent ECT when administered with succinylcholine without worsening muscle disease. 37 , 63 , 64 Ntatsaki et al. used the nondepolarizing muscle relaxant mivacurium 0.2 mg/kg followed by reversal with neostigmine and glycopyrrolate in a patient with inclusion myositis. 65 Management strategies in inflammatory myopathies include monitoring CK levels and providing enhanced anesthetic care to prevent aspiration. Section title: Bone fractures, muscle tears, and vascular injuries Educational score: 3.9019174575805664 Domain: biomedical Document type: Review Language: en In the preanesthetic era of ECT, musculoskeletal injuries were expected during convulsive movements. In modern practice, fractures are not necessarily contraindications for ECT. Case series suggest that ECT can be safely and successfully given to patients with bone fractures with coordinated care from orthopedic surgeons and anesthetists. 66 , 67 Similarly, ECT was safely administered 2 weeks after the repair of the severed strap muscle and the anterior jugular vein. 68 Precaution is required in such conditions; succinylcholine in an adequate dose (1.5 mg/kg) may bring sufficient muscle relaxation, protecting musculoskeletal structures. There were cases of successful ECT with no adverse events in patients with Harrington rod implants. 69 Section title: Implanted devices Educational score: 4.288593292236328 Domain: biomedical Document type: Review Language: en (a) Pacemakers Pacemakers or implanted defibrillators are not contraindications for ECT. 70 , 71 , 72 Supraventricular tachycardia was reported in one patient, which resolved promptly and did not prevent further ECT. The synchronous mode is preserved as serious arrhythmias are a risk of the asynchronous mode. A cardiologist's opinion must be sought before proceeding to ECT for patients with these devices. (b) Deep brain stimulators A review of 21 cases showed that ECT was safe with deep brain stimulators, provided precautions were taken. 73 Burr holes through which deep brain simulators (DBSs) were placed must be avoided during ECT, and the device is to be reprogrammed to 0 V and switched off. Although the DBS was turned off during the whole ECT course in seven instances, it was only turned off immediately before ECT in other cases and turned on in recovery units. There was no evidence of brain tissue damage, a change in neurological function, DBS device damage, or reprogramming because of ECT. In studies where DBS electrode placement was confirmed before and after ECT by neuroimaging, there was no change in the position of the DBS electrodes. (c) Cochlear implant A cochlear implant was previously considered an absolute contraindication for ECT. 74 As years passed, it was found that cochlear implant users needed ECT. Reflecting on this need, the Danish Psychiatric Association and other authors reviewed the literature and later concluded that a cochlear implant is not an absolute contraindication. 75 ECT did not damage the implant even with the highest level of energy delivered from an ECT device. 75 Stimulations in all but one patient were administered through the side contralateral to the implant; for one patient who had the implant fitted to the right ear, right unilateral ECT was administered. 76 Discomfort around the jaw was the only reported adverse effect. However, given the dearth of data about ipsilateral ECT with the cochlear implant, it is safe to administer stimulations at the maximum distance away from the implant. Bifrontal ECT is safe in this regard. Section title: Pregnancy Educational score: 4.178079605102539 Domain: biomedical Document type: Review Language: en The official joint statement from the American Psychiatric Association and the American College of Obstetricians and Gynecologists, several original studies, and meta‐reviews indicate that ECT is usually a safe and effective treatment in pregnancy. 77 , 78 , 79 However, some authors published a contradictory report and cautioned that ECT should be used as a last resort in pregnancy. 80 The findings included a reduced fetal heart rate and several fetal deaths (7.1% from ECT given to 169 pregnant women). 80 In one instance, fetal death occurred after status epilepticus in the mother following ECT. 81 Other adverse events were fetal arrhythmias, premature birth, preeclampsia, vaginal bleeding, and uterine contractions without labor. Although there are inconsistent views, most reviews are reassuring, and reports differ due to varied attributions of adverse events to ECT. 77 , 78 , 79 A recent review rated complications as low‐to‐moderate grade and not life‐threatening to pregnant women. 82 Joint management with the obstetric team involving frequent fetal monitoring is critical. A semi‐prone position, endotracheal intubation to reduce the risk of aspiration, the elevation of the right hip to improve the placental perfusion, extended fetal monitoring, and inhalational anesthetics to minimize the risk of uterine contractions are essential considerations for ECT in pregnancy. 83 There are few reports on ECT during special situations like twin pregnancies where there were no adverse outcomes. 84 , 85 Section title: Epilepsy Educational score: 2.7075188159942627 Domain: biomedical Document type: Other Language: en ECT has an anticonvulsant effect and is hence not contraindicated in epilepsy or if there is a history of previous seizures. 86 However, a neurological evaluation is to be completed to determine the cause of the seizure, which may imply extra caution for ECT. Section title: ECT and concomitant medications Educational score: 4.20745849609375 Domain: biomedical Document type: Study Language: en (a) Theophylline Co‐administration of ECT and theophylline medications was followed by prolonged and recurrent seizures in one patient and prolonged seizure (190 s) in another. 87 , 88 Theophylline is known to induce seizure at a serum level of 25–50 μg/mL. However, recurrent seizure after ECT occurred at 22.6 μg/dL, measured 3 h after ECT, suggesting an interaction. 87 It is therefore crucial to measure the theophylline level before ECT and ensure it is well below seizure‐prone levels. In most documented cases, ECT was uneventful when administered with theophylline. 89 Interestingly, theophylline with a level of 10–15 μg/mL was also used to induce or generate sufficiently long seizures for therapeutic purposes. 89 (b) Lithium Earlier case reports and retrospective chart reviews showed prolonged seizure, apnea, and delirium with concomitant lithium and ECT. 90 , 91 , 92 However, a recent prospective randomized controlled trial and a retrospective study did not observe severe complications because of this combination when lithium levels were below 0.6 mEq/L. 93 , 94 Caution is still required, and lithium must be withheld before ECT in the morning. (c) Antiepileptics Contrary to the conventional wisdom, several reports including controlled trials showed that antiepileptic medications did not affect seizure quality or the clinical efficacy of ECT. 95 , 96 , 97 Therefore, these medications may be continued during ECT. Although results of combined ECT and benzodiazepines are mixed, the overall picture suggests that seizure can be achieved with clinical efficacy, especially in catatonia, for which this combination is highly useful. 97 Stimulus dose titration to determine the seizure threshold is recommended when ECT is given with antiepileptics (Table 2 ). Section title: Other conditions Educational score: 4.352262020111084 Domain: biomedical Document type: Review Language: en (a) Kidney diseases and pheochromocytoma Individuals with renal failure undergoing hemodialysis received ECT without adverse reactions. 106 , 107 , 108 Caution is required to reduce BP before administering ECT with β‐blockers such as esmolol. 107 Pheochromocytoma is a rare neoplasm with an incidence of 0.2–0.8 per 100,000 individuals. 109 Hypertension can occur in this condition because of the catecholamine‐secreting tumor. A 34‐year‐old woman with pheochromocytoma died 3 h after ECT due to sudden vascular collapse. 110 The authors of this report proposed that death could be attributed to ECT, vascular collapse from sodium thiopental, or emotional stress. Notably, the pulse rate was 100/min, and there was no record of hypertension. In another instance, a 27‐year‐old man with pheochromocytoma received ECT. In minutes after the first stimulation, the systolic BP went up to 260 mm Hg, which was reduced by administering a β‐blocker. The patient had subsequent ECT without complications. 111 (b) Ophthalmic conditions An increase in intraocular pressure (IOP) occurs immediately after the beginning of the seizure, which returns to normal ∼3 min after stimulation. 98 While retinal detachment was reported during the recovery from bilateral ECT in one instance, no adverse ophthalmological outcomes were noted after ECT in patients who had previous retinal detachment, albeit 30 years ago in one case. 99 , 100 , 107 No ocular complications occurred in relation to ECT after cataract surgery, in glaucomatous eyes, or in the presence of titanium‐based trabecular microbypass stents for open‐angle glaucoma treatment. 102 , 103 , 112 There is a need for regular ophthalmic consultation and IOP measurements when a patient presents with such risk factors. (c) Respiratory diseases Considering that ECT involves general anesthesia and assisted ventilation, careful consideration of pulmonary conditions is crucial. According to one retrospective study, four of 34 patients with asthma experienced an exacerbation of symptoms, which were successfully managed by standard anti‐asthma treatments. 113 However, no complications were reported after ECT in patients with chronic obstructive pulmonary disease. 114 As mentioned earlier in this review, the use of theophylline should be carefully evaluated. (d) Fever and infection A single case report showed the safety and efficacy of ECT in a 4‐year‐old boy with febrile infection‐related epilepsy syndrome (FIRES) that followed pharyngitis and high fever. 115 ECT was successfully given to patients with symptomatic COVID‐19. 116 In the Regestein and Reich case series, three out of 19 patients who received ECT had signs of infection, including Gram‐positive septicemia and unrelenting fever. 15 Tachycardia was observed, with the highest reported pulse rate of 180/min. Death was reported in a young patient with massive PE, pleural effusion, and staphylococcal pneumonia after developing ventricular fibrillation during ECT. 15 (e) Obesity and conditions that increase the risk of aspiration Obesity can pose challenges during ECT, given delayed gastric and esophageal emptying and difficulties in protecting the airway. 104 , 117 Also, prospective observational data suggest that obesity and the duration of seizure were the factors correlated with post‐ECT desaturation. 118 However, the duration of unconsciousness during ECT is relatively brief. A review of 50 obese patients with a body mass index (BMI) of 45 or above who received 660 ECT sessions demonstrated a safe administration of the treatment without a requirement for tracheal intubation. 104 Patients fasted overnight and received 100% oxygen with assistance of positive pressure using a face mask. ECT was performed in the supine position with a 15–30° elevation of the upper body. In another instance, when hypoxic episodes occurred because of suspected inadequate ventilation, a lower dose of succinylcholine, 0.5 mg/kg based on the total body weight, helped to restore adequate oxygenation. 119 This may be understood against a background of increased muscular consumption of oxygen because of succinylcholine‐induced fasciculations. 120 Some authors, therefore, recommend nondepolarizing agents, such as rocuronium, in obesity. 105 Other strategies to reduce the risk of adverse outcomes with ECT in obesity include a supraglottic device and noninvasive positive pressure ventilation. Section title: Age Educational score: 3.347580909729004 Domain: biomedical Document type: Study Language: en Although advanced age is associated with increased medical morbidities, old age per se is not associated with mortality after ECT. 121 Until now, the oldest person to receive ECT was a 100‐year‐old woman who safely underwent the treatment. 40 ECT is safe in children. 122 , 123 No evidence supports the fear that ECT adversely affects brain growth (Table 3 ). Section title: LIMITATIONS Educational score: 3.5190091133117676 Domain: biomedical Document type: Review Language: en The above results are to be cautiously interpreted. They are from limited evidence constituted by case reports and case series, uncontrolled data, and retrospective reviews. The reproducibility of some of these results is thus limited. Also, this report is prepared based on extant literature in which serious and fatal outcomes may not be reported. Still, ECT is a time‐tested intervention in psychiatry and has remained in practice for over eight decades. This narrative review is biased toward a nonsystematic selection of the literature without a specific protocol and hence is not inclusive of every report of ECT in one or another medical condition. Section title: CONCLUSION Educational score: 3.8218486309051514 Domain: biomedical Document type: Other Language: en In general, ECT can be safely administered to patients with various medical conditions and physiological complexities. In certain conditions, ECT may be avoided or administered with high caution if indicated based on a favorable risk–benefit ratio as relevant for individual patients. These conditions include a recent myocardial infarction in the last 4 weeks, intracranial aneurysms of more than 1 cm in size, proximal DVT, pheochromocytoma, and systemic infections. In particular, fatalities associated with DVT warrant extreme precaution and monitoring in this condition. In a nutshell, the extension of ECT to patients with medical and physiological complexities expands the scope of this treatment and thus the horizon of psychiatry. Section title: AUTHOR CONTRIBUTIONS Educational score: 0.932667076587677 Domain: other Document type: Other Language: en Alby Elias, Soumitra Das, Sarabjit Loyal, and Naveen Thomas contributed to the conceptualization and design of the study. Data acquisition and analysis were performed by Alby Elias, Soumitra Das, and James Kirkland. Naveen Thomas, Sarabjit Loyal, and Soumitra Das contributed significantly to drafting the manuscript and developing the figures and tables. Section title: CONFLICT OF INTEREST STATEMENT Educational score: 0.8662789463996887 Domain: other Document type: Other Language: en The authors declare no conflicts of interest. Section title: ETHICS APPROVAL STATEMENT Educational score: 0.9318303465843201 Domain: other Document type: Other Language: en The ethics approval statement is not applicable. Section title: PATIENT CONSENT STATEMENT Educational score: 1.0576378107070923 Domain: other Document type: Other Language: en The patient consent statement is not applicable. Section title: CLINICAL TRIAL REGISTRATION Educational score: 1.071506381034851 Domain: biomedical Document type: Other Language: en The clinical trial registration is not applicable. | Review | biomedical | en | 0.999997 |
PMC11695779 | Section title: Introduction Educational score: 4.569487571716309 Domain: biomedical Document type: Study Language: en Central precocious puberty (CPP) is a common pediatric endocrine disorder characterized by the premature activation of the hypothalamic-pituitary-gonadal (HPG) axis, resulting in the development of secondary sexual characteristics before the age of 8 years in girls and 9 years in boys. This pediatric endocrine disorder exhibits a notable gender disparity in its prevalence, with a significantly higher incidence in girls. Epidemiological data suggest that the prevalence ranges from 0.217 to 26.28 per 10,000 in girls, compared to a much lower range of 0.02 to 0.9 per 10,000 in boys ( 1 ). This gender disparity suggests that genetic factors may play a critical role in the pathogenesis of CPP. Although the precise molecular mechanisms remain under investigation, genetic contributions are emerging as a key area of research in understanding this condition. Previous studies have identified the makorin ring-finger protein 3 ( MKRN3 ) gene as a crucial factor in the onset of puberty. MKRN3 negatively regulates the HPG axis, and loss-of-function (LOF) mutations in this gene can lead to increased gonadotropin-releasing hormone (GnRH) secretion, premature activation of the HPG axis, and subsequent development of CPP ( 2 – 4 ). Section title: Introduction Educational score: 4.549026012420654 Domain: biomedical Document type: Study Language: en The MKRN3 gene, which encodes a RING (really interesting new gene) zinc finger protein, was first identified in 1999 by Jong MT et al. during their investigation into the molecular mechanisms of Prader-Willi syndrome ( 5 ). Located on chromosome 15q11.2, a critical region associated with Prader-Willi syndrome, MKRN3 is a single exon, paternally expressed imprinted gene that encodes an E3 ubiquitin ligase. MKRN3 protein contains multiple domains, including two C3H motifs at the N-terminus, followed by a unique makorin-type Cys-His (CH)-rich domain, a C3H4 RING zinc finger, and a terminal C3H motif. The RING zinc finger domain is responsible for its E3 ubiquitin ligase activity, while the C3H zinc finger motifs are involved in RNA binding. MKRN3 plays a role in protein degradation and is involved in various cellular processes, including signal transduction, cell cycle regulation, differentiation, and morphogenesis ( 6 ). Section title: Introduction Educational score: 3.4251649379730225 Domain: biomedical Document type: Clinical case Language: en While numerous studies have explored the genetic underpinnings of CPP, data from the Chinese population remains limited. This report presents a case of CPP in a Chinese child attributed to a novel MKRN3 gene mutation, aiming to enhance clinical understanding of this condition and its genetic determinants. Section title: General information Educational score: 2.9117696285247803 Domain: clinical Document type: Clinical case Language: en A 4-year-9-month-old female patient presented to our hospital on March 17, 2021, with a chief complaint of “breast development for 2 months”. At birth, a congenital melanocytic nevus measuring approximately 1.5 cm × 2 cm with irregular borders was noted on her right waist. At 4 years and 7 months of age, the patient experienced thelarche, which spontaneously regressed without medical intervention. However, two weeks prior to this admission, the patient again noticed recurrent breast development. Section title: Personal history Educational score: 2.940882444381714 Domain: clinical Document type: Clinical case Language: en The patient is the firstborn from an uncomplicated, full-term cesarean section delivery, with no history of perinatal complications. Birth weight was 3.4 kg, and birth length was 50 cm. Developmental milestones, including gross motor skills and social interaction, were appropriate for age. The maternal prenatal course was unremarkable. There is no history of consanguinity between the parents. The father, with a height of 167 cm, experienced his pubertal growth spurt at approximately 12 years of age, with growth cessation at 14 years. The mother, with a height of 162 cm, experienced menarche at 13 years of age. Section title: Physical examination Educational score: 3.689396619796753 Domain: clinical Document type: Clinical case Language: en The patient’s height was 116 cm, which is slightly above the average for her age (109 ± 4.1 cm), with a weight of 20 kg and a BMI of 14.9, all in the normal range (17.8 ± 2.4 kg and 15.3 ± 1.6). No dysmorphic facial features were observed. A 3 cm × 4 cm irregular café-au-lait macule was present on the right waist. There was no evidence of acne or dorsocervical fat pad. The thyroid gland was non-palpable. Breast development was consistent with Tanner stage II, with bilateral breast buds measuring approximately 1.5 cm × 1.5 cm, and no areolar hyperpigmentation. The external genitalia were age-appropriate, with the labia majora fully covering the labia minora. Pubic hair development was consistent with Tanner stage I, and no axillary hair was present. The spine and limbs showed no deformities, and the neurological examination was unremarkable. Section title: Laboratory investigations Educational score: 4.141049861907959 Domain: biomedical Document type: Study Language: en Laboratory tests revealed a normal complete blood count, comprehensive metabolic panel, and urinalysis. Tumor markers, including alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and carbohydrate antigen 19-9 (CA 19-9), were within normal limits. Thyroid function tests, including thyroid-stimulating hormone (TSH) at 1.75uIU/mL (reference range:0.4-6uIU/mL), free thyroxine (free T4) at 17.24pmol/L (reference range:8.37-29.6pmol/L), free triiodothyronine (free T3) at 6.96pmol/L (reference range:2.5-9 pmol/L) were all within normal ranges. Insulin-like growth factor 1 (IGF-1) at 262 ng/mL (reference range: 49-283ng/mL) was normal. Adrenocorticotropic hormone (ACTH) was measured at 13.92 pg/mL (reference range: 0 - 46 pg/mL), and cortisol was within the normal range at 273.30 nmol/L (reference range: 138.2 - 691 nmol/L). Section title: Laboratory investigations Educational score: 4.025722026824951 Domain: biomedical Document type: Study Language: en Sex hormone analysis showed the following levels: luteinizing hormone (LH) at 0.3 IU/L (reference range: 0 - 0.33 IU/L), follicle-stimulating hormone (FSH) at 2.14 IU/L (reference range: 0.42 - 5.45 IU/L), prolactin at 7.64 ng/mL (reference range: 4.2 - 23.04 ng/mL), estradiol at 15.08 pg/mL (reference range: 0 - 10 pg/mL), total testosterone at 12.64 ng/dL (reference range: 1.1 - 62 ng/dL), and progesterone at 0.21 ng/mL (reference range: 0 - 0.35 ng/mL), as shown in Table 1 . Section title: Imaging studies Educational score: 3.0912668704986572 Domain: biomedical Document type: Clinical case Language: en Breast ultrasound revealed bilateral breast buds with the left breast measuring 2.9 cm × 0.7 cm × 3.4 cm and the right breast measuring 2.9 cm × 0.6 cm × 2.9 cm. The margins were ill-defined, with no significant internal abnormal echogenicity or increased vascularity detected. Section title: Imaging studies Educational score: 3.355546236038208 Domain: biomedical Document type: Clinical case Language: en Pelvic ultrasound showed a uterus measuring 3.0 cm × 1.5 cm × 0.9 cm with a thin endometrial stripe. The left ovary measured 2.4 cm × 0.7 cm × 1.0 cm, containing two follicles each measuring 0.3 cm, while the right ovary measured 2.9 cm × 1.0 cm × 1.2 cm, containing six follicles each measuring 0.3 cm. Section title: Imaging studies Educational score: 2.1790592670440674 Domain: biomedical Document type: Clinical case Language: en Abdominal ultrasound showed no evidence of adrenal masses or enlargement. Brain MRI demonstrated a normal-appearing pituitary gland. Section title: Genetic analysis Educational score: 1.169230580329895 Domain: biomedical Document type: Other Language: en All clinical investigations and therapeutic interventions were performed with informed consent from the patient’s legal guardians and were approved by the institutional ethics committee. Section title: Genetic analysis Educational score: 2.6366186141967773 Domain: biomedical Document type: Study Language: en After obtaining informed consents, 3 mL of EDTA-anticoagulated peripheral blood was collected from the patient and her parents. High-throughput whole exome sequencing (WES) was performed by Beijing Maikeno Technology Co., Ltd. Section title: Genetic analysis Educational score: 4.193423271179199 Domain: biomedical Document type: Study Language: en Briefly, genomic DNA was extracted from the blood samples and subjected to DNA library preparation. The library was then hybridized with biotinylated probes targeting the whole exome (P039-Exom, Maikeno) under optimized conditions. Streptavidin-coated magnetic beads were utilized to capture the biotinylated probe-target DNA complexes. The captured DNA fragments were then eluted, purified, and enriched. The enriched target DNA was sequenced using the Illumina NextSeq 500 platform. Sequencing reads were aligned to the human reference genome hg19 using the Burrows-Wheeler Aligner (BWA) software. The resulting alignment files were sorted, filtered, and locally realigned to reduce false-positive variant calls. Variants of potential clinical significance were confirmed by Sanger sequencing. Section title: Genetic sequencing results Educational score: 4.330164909362793 Domain: biomedical Document type: Study Language: en A heterozygous frameshift mutation was identified in exon 1 of the MKRN3 gene in the proband . This single nucleotide deletion causes a frameshift that leads to the substitution of arginine (R) at codon 407 with glycine (G), ultimately resulting in premature termination of protein translation 73 amino acids downstream (p.R407Gfs*75). Familial segregation analysis confirmed that the proband’s father is a heterozygous carrier of the mutation, while the mother carries the wild-type (WT) allele at this locus . Section title: Genetic sequencing results Educational score: 2.9531681537628174 Domain: biomedical Document type: Other Language: en This variant is absent from population databases and has not been previously reported in the Human Gene Mutation Database (HGMD). Additionally, ClinVar does not provide pathogenicity assessments for this variant. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, the variant is classified as likely pathogenic. Section title: Uniqueness of the case and discovery of a novel MKRN3 mutation Educational score: 3.971808433532715 Domain: biomedical Document type: Clinical case Language: en In this study, we report a case of a 4-year-9-month-old Chinese girl diagnosed with ICPP caused by a novel heterozygous frameshift mutation in the MKRN3 gene . This mutation leads to premature termination of the protein, resulting in a loss-of-function protein. The discovery of this mutation expands the known pathogenic mutation spectrum of the MKRN3 gene, particularly in the Chinese population where data on MKRN3 mutations are relatively scarce. This case provides a foundation for further exploration of the genetic characteristics and pathogenic mechanisms of MKRN3 in the Chinese population. Section title: Further insights into the pathogenesis of CPP Educational score: 4.4445390701293945 Domain: biomedical Document type: Study Language: en Previous studies have shown that the MKRN3 gene plays an inhibitory role in suppressing GnRH secretion before puberty ( 7 ). In 2013, Abreu et al. pioneered the identification of pathogenic mutations in the MKRN3 gene as a causative gene for CPP through WES analysis of 40 affected individuals from 15 families ( 8 ). They specifically identified four distinct MKRN3 variants in 12 CPP patients across 5 families, including one missense and three frameshift mutations ( 8 ). Subsequent research across diverse populations has further expanded the repertoire of pathogenic MKRN3 variants to a total of 54, with these mutations primarily classified as frameshift, missense, or nonsense ( 9 – 12 ) ( Table 2 ). The novel mutation identified in this study is not located in the last base pairs, the mutated mRNA probable undergoes a nonsense mediated decay (NMD), and we consider that this novel discovered mutation would be a loss-of-function mutation. Although no experimental structural data is available for MKRN3 protein, the three-dimensional (3D) structure predicted by AlphaFold suggests that the R407G mutation occurs in the c-terminal C3H domain . This variant may retain E3 ubiquitin ligase activity but likely lacks RNA-binding capability due to the disruption of the C3H motif. Section title: Further insights into the pathogenesis of CPP Educational score: 4.8591766357421875 Domain: biomedical Document type: Study Language: en The exact role of MKRN3 in regulating the central neuroendocrine system and how its mutations lead to CPP is still unclear. Studies in animals show that MKRN3 can inhibit the secretion of gonadotropin-releasing hormone 1 (GnRH1). In mice, MKRN3 mRNA levels peak around postnatal days 10-12 and then drop sharply by day 15, aligning with the start of puberty. Under normal conditions, MKRN3 ubiquitinates methyl-CpG binding domain protein 3 (MBD3), disrupting its binding to the GnRH1 promoter. This prevents the recruitment of TET2, leading to increased promoter methylation and suppression of GnRH1 transcription. In humans, serum MKRN3 levels were found to exhibit substantial inter-individual variability but consistently decline prior to the onset of puberty, showing a negative correlation with gonadotropin secretion. This observation suggests that MKRN3 may exert an inhibitory effect on hypothalamic GnRH secretion during childhood ( 13 ). Decreased MKRN3 levels during puberty could lead to the release from suppression of the HPG axis, thereby triggering pubertal development ( 14 ). Moreover, some children with CPP exhibit markedly reduced or undetectable serum MKRN3 levels, potentially linked to MKRN3 gene mutations ( 14 ). It is thus hypothesized that LOF mutations in MKRN3 could compromise its inhibitory function over the HPG axis, leading to pulsatile GnRH release, premature activation of the axis, and consequently, precocious puberty. Further investigations into the relationship between MKRN3 and sex hormones have revealed an inverse correlation between MKRN3 levels and peripheral blood LH, FSH, and estradiol (E2) levels, but not with anti-Müllerian hormone (AMH) levels ( 15 ). These findings reinforce the concept that MKRN3 acts as a negative regulator of gonadotropins and E2, suppressing hypothalamic GnRH secretion during childhood and declining prior to pubertal onset. Elucidating the expression patterns and functional roles of MKRN3 protein is crucial for understanding the regulatory mechanisms underlying human pubertal initiation. Section title: Clinical management implications of this case Educational score: 4.101725101470947 Domain: biomedical Document type: Study Language: en The mutation in this patient was inherited from her asymptomatic father, consistent with the imprinting inheritance pattern of the MKRN3 gene. Since MKRN3 is a paternally expressed imprinted gene, mutations are typically passed from asymptomatic fathers to their offspring. Understanding the family’s genetic background, especially the developmental history of the father, is crucial for accurate diagnosis and family genetic counseling. This study highlights the importance of comprehensive genetic analysis in early-onset ICPP cases to accurately identify pathogenic mutations. Section title: Clinical management implications of this case Educational score: 3.9022862911224365 Domain: biomedical Document type: Other Language: en Identifying the genetic cause of CPP early is crucial for personalized treatment. The mutation in this patient has not been reported in public databases, indicating that it is a novel pathogenic variant. For cases like this, clinicians should consider early genetic testing to formulate personalized management strategies. Moreover, as CPP caused by MKRN3 mutations tends to have an earlier onset, close monitoring and follow-up are necessary, especially for asymptomatic individuals from the same family, particularly paternal family members, to ensure timely intervention. Section title: Therapeutic and prognostic outlook Educational score: 3.9707255363464355 Domain: biomedical Document type: Review Language: en Currently, GnRH analogs are the standard treatment for CPP. These analogs exert their therapeutic effect primarily at the anterior pituitary, where they competitively bind to GnRH receptors on gonadotrophs. While an initial transient increase in LH, FSH, and sex hormones may be observed, continuous administration leads to pituitary desensitization, reducing gonadotropin secretion and subsequently suppressing sex hormone levels, thus effectively interrupting the prematurely activated HPG axis. Section title: Therapeutic and prognostic outlook Educational score: 3.965214252471924 Domain: biomedical Document type: Study Language: en Importantly, extended GnRH analog therapy has been recommended for individuals with CPP caused by MKRN3 gene mutations ( 16 ). While the therapeutic response to GnRH analogs in MKRN3-induced CPP is generally comparable to that observed in idiopathic CPP, the earlier average age of onset in MKRN3-related cases necessitates vigilant monitoring of even asymptomatic children from affected families. This proactive approach ensures the timely identification of CPP onset and allows for prompt intervention, optimizing treatment outcomes. Section title: Therapeutic and prognostic outlook Educational score: 3.9956164360046387 Domain: biomedical Document type: Study Language: en Although GnRH analogs remain the primary treatment for CPP, long-term efficacy and therapeutic strategies for CPP caused by MKRN3 mutations may need further optimization. Since early intervention is critical in preventing premature epiphyseal closure and limiting adult height, this study supports the importance of early genetic screening and individualized treatment for such cases. Section title: Conclusions Educational score: 4.034976959228516 Domain: biomedical Document type: Study Language: en By linking this case to the specific findings, this study not only adds to the body of evidence on the genetic basis of CPP but also provides new insights and directions for future clinical management and research. This study underscores the importance of MKRN3 mutations in the Chinese population, but further functional studies are needed to elucidate the precise pathophysiological mechanisms. These studies will help improve our understanding of the genetic mechanisms underlying CPP and lead to more precise therapeutic approaches. | Study | biomedical | en | 0.999998 |
PMC11695781 | Section title: 1 Introduction Educational score: 1.1073943376541138 Domain: other Document type: Other Language: en While reinforcement learning (RL) has gained popularity in policy learning, many problems that require coordination and interaction between multiple agents cannot be formulated as single-agent reinforcement learning. Examples of such scenarios include self-driving cars , autonomous intersection management , multiplayer games , and distributed logistics . Solving these kinds of problems using single-agent RL is problematic because the interaction between agents and the non-stationary nature of the environment due to multiple learning agents can not be considered . Multi-agent reinforcement learning (MARL) and cooperative learning between several interacting agents can be beneficial in such domains and has been extensively studied . Section title: 1 Introduction Educational score: 1.289014220237732 Domain: other Document type: Other Language: en However, when several agents are interacting with each other in an environment without real-time communication, the lack of communication deteriorates policy learning. To alleviate this problem, we propose a meta-trajectory-based communication scheme during training, where agents indirectly share information through a centralized critic. By aggregating trajectories from all agents into a meta-trajectory, the critic is able to learn the cooperative dynamics between agents, even in the absence of direct communication during execution. This scheme enables the agents to implicitly learn from each other’s observations and rewards, allowing them to better predict each other’s behavior and adapt accordingly. For example, in applications like autonomous driving at intersections, agents can anticipate the actions of others, improving performance, safety, and cooperation. Section title: 1 Introduction Educational score: 1.631280541419983 Domain: other Document type: Other Language: en A standard paradigm for multi-agent planning is to use the centralized training and decentralized execution (CTDE) approach , which we also adopt in this work. During centralized training, the critic receives global information, including observations and actions from all agents, allowing it to model the inter-agent dynamics. During decentralized execution, each agent uses its own local observations to act independently. Section title: 1 Introduction Educational score: 3.032691478729248 Domain: other Document type: Study Language: en In this work, we propose a new cooperative multi-agent reinforcement learning algorithm, which is an extension to Proximal Policy Optimization (PPO), called Multi-Agent Cooperative Recurrent Proximal Policy Optimization (MACRPO). MACRPO enhances inter-agent coordination through two key mechanisms: First, it uses a recurrent long short-term memory (LSTM) layer in the critic network, trained with a meta-trajectory that combines trajectories from all agents . This enables the critic to capture the temporal dynamics and interactions between agents over time, while also handling the partial observability of each agent. Second, MACRPO introduces a novel advantage function estimator that incorporates both the rewards and value functions of other agents, controlled by a cooperation parameter. This allows MACRPO to adjust the level of cooperation, which is particularly useful in environments where agents cannot fully cooperate, balancing individual and collective rewards. Section title: 1 Introduction Educational score: 1.3145642280578613 Domain: other Document type: Other Language: en MACRPO operates under the centralized training and decentralized execution paradigm. During training, the centralized critic leverages the meta-trajectory to sequentially predict the value of a state for each agent, enabling it to learn the cooperative strategies between agents. During execution, the decentralized actor networks use only local observations, ensuring that each agent acts autonomously without requiring real-time communication. Section title: 1 Introduction Educational score: 1.133411169052124 Domain: other Document type: Other Language: en Moreover, in environments where multiple agents are simultaneously learning during training, each agent’s policy and the environment’s dynamics are constantly changing from the perspective of other agents, resulting in non-stationarity . To mitigate this issue, MACRPO employs an on-policy approach, ensuring that the most recent data collected from the environment is used for training. Section title: 1 Introduction Educational score: 2.463531732559204 Domain: other Document type: Study Language: en In summary, our contributions are as follows: (1) We propose a cooperative on-policy centralized training and decentralized execution framework that is applicable to both discrete and continuous action spaces. (2) We introduce two novel mechanisms for information sharing across agents: (a) a recurrent LSTM component in the network architecture that integrates meta-trajectories to capture inter-agent cooperation over time, and (b) an advantage function estimator that combines individual rewards and value functions with a control parameter, which allows the level of cooperation between agents to be dynamically adjusted. This dual mechanism enables superior policy learning and adaptability compared to prior approaches, as demonstrated in our experiments. (3) We evaluate MACRPO on three cooperative multi-agent tasks: DeepDrive-Zero , Multi-Walker , and Particle , showing that it achieves comparable or superior performance against state-of-the-art methods. Section title: 1 Introduction Educational score: 1.4007757902145386 Domain: other Document type: Other Language: en The rest of this paper is organized as follows. The review of related works in Section 2 demonstrates that while MARL has been extensively studied, existing approaches do not address the dynamics of interaction between agents in detail. In Section 3 , we provide the required background in Markov Games and Proximal Policy Optimization. The problem definition and the proposed method are described in Section 4 , with emphasis on the two innovations, meta-trajectory for recurrent network training and joint advantage function. Then, Section 5 presents empirical evaluation in three multi-agent environments showing superior performance of the proposed approach compared to the state-of-the-art. Finally, in Section 6 we conclude that implicit information sharing can be used to improve cooperation between agents while discussing its limitations in settings with a high number of agents. Section title: 2 Related work Educational score: 1.5784317255020142 Domain: other Document type: Study Language: en The most straightforward and maybe the most popular approach to solving multi-agent tasks is to use single-agent RL and consider several independent learning agents. Some prior works compared the performance of cooperative agents to independent agents and tried independent Q-learning and PPO with LSTM layer , but they did not work well in practice . Also, Zhao et al. tried to learn a joint value function for two agents and used PPO with an LSTM layer to improve the performance in a multi-agent setting. Section title: 2 Related work Educational score: 1.6418691873550415 Domain: other Document type: Study Language: en In order to use single-agent RL methods for multi-agent settings, improve the performance, and speed up the learning procedure, some works used parameter sharing between agents . Especially in self-play games, it is common to use the current or older versions of the policy for other agents . We will compare our proposed method with several state-of-the-art single-agent RL approaches with shared parameters between agents proposed in Terry et al. in the experiments section. Our way of training the LSTM layer in the critic differs from parameter sharing used in the literature such that instead of using separate LSTMs for each agent, the LSTM layer in our method has a shared hidden state, which is updated using a combination of all agents’ information. This lets the LSTM layer learn about the dynamics of interaction and cooperation between agents across time. Section title: 2 Related work Educational score: 2.8212642669677734 Domain: other Document type: Study Language: en In addition to using single-agent RL methods with or without parameter sharing, some works focus on designing multi-agent RL algorithms that incorporate communication between agents to enhance coordination in multi-agent settings. Communication in multi-agent environments can significantly improve learning and performance by enabling agents to share information . However, the effectiveness of communication often depends on the availability and optimization of the communication channels. IC3Net introduces a communication gating mechanism, allowing agents to learn when to communicate with each other during cooperative and competitive tasks. While IC3Net leverages communication between agents to improve coordination, it assumes that a communication channel is available during execution. In some real-world scenarios, such as autonomous driving, direct communication between agents may not be feasible. In such environments, agents must learn to cooperate without explicit communication. Unlike IC3Net, MACRPO addresses this challenge by enabling indirect information sharing during the training phase through a meta-trajectory that combines the experiences of all agents. This meta-trajectory allows agents to implicitly learn cooperation strategies without requiring explicit communication during execution, making MACRPO well-suited for environments where communication is restricted or absent. Section title: 2 Related work Educational score: 1.869588017463684 Domain: other Document type: Study Language: en A recently popularized paradigm for sharing information between agents is the use of centralized training and decentralized execution. In general, we can categorize these types of approaches into two groups: value-based and actor-critic-based. In value-based methods, the idea is to train a centralized value function and then extract the value functions for each agent from that to act in a decentralized manner in the execution time . On the other hand, the actor-critic-based methods have actor and critic networks . The critic network has access to data from all agents and is trained in a centralized way, but the actors have only access to their local information. They can act independently in the execution time. The actors can be independent with individual weights or share the policy with shared weights . In this work, we use an actor-critic-based method with centralized training and decentralized execution, providing two innovations to improve information sharing without a communication channel between agents during execution. Section title: 2 Related work Educational score: 3.3961832523345947 Domain: other Document type: Study Language: en RMAPPO is a method close to ours, which uses the CTDE framework. They make no mention of recurrent neural networks (RNNs) in their paper, but their code contains recurrent layers. RMAPPO focuses primarily on adapting PPO components such as clipping, mini-batching, batch size, value normalization, and value function input representation for multi-agent environments. However, the main distinction between our work and theirs lies in the meta-trajectory we generate from the data of all agents, and the specific manner in which we employ the RNN layer. RMAPPO employs CTDE and RNNs as usual without a combined trajectory as input, which limits its ability to model interactions between agents over time. In addition to the meta-trajectory, another difference is in the way information is shared. While both methods share policy parameters across agents, RMAPPO uses a shared reward function for all agents that is the sum of individual rewards, without any control over the degree of cooperation. In contrast, MACRPO introduces a novel advantage function estimator that incorporates a control parameter, allowing us to dynamically adjust the level of cooperation between agents. This provides greater flexibility in environments where the degree of cooperation must vary dynamically over time. Furthermore, while RMAPPO is tested primarily in environments with discrete action spaces, MACRPO is evaluated in both discrete and continuous action spaces, demonstrating its broader applicability. Section title: 2 Related work Educational score: 1.500348448753357 Domain: other Document type: Study Language: en In Foerster et al. , which is another work near ours, the actor is recurrent, but the critic is a feed-forward network, whereas our actor and critic are both recurrent, and the recurrent layer in our critic has a crucial role in our method. Their method is also for settings with discrete action spaces, whereas we test our method on three environments with both discrete and continuous action spaces. Section title: 2 Related work Educational score: 1.7842384576797485 Domain: other Document type: Other Language: en ROLA is another work near ours. They use LSTMs in both actor and critic networks. Additionally, ROLA employs both centralized and individual asymmetric critics that estimate individual advantage values using local history and/or state information. However, we construct the meta-trajectory which has not only the history of each agent but also the history of the interaction between agents and the environment’s dynamics. In addition, we propose a novel advantage function estimator which is a combination of all agents’ advantage functions and the cooperation level of agents can be changed based on the problem using a control parameter. Section title: 2 Related work Educational score: 1.9886544942855835 Domain: other Document type: Study Language: en Durugkar et al. is also a work that combines an agent-specific reward and an environment-specific reward to accomplish the shared task. They consider a framework that uses a linear mixing scheme to balance individual preferences and task rewards. They demonstrate that in their test environments, a small amount of selfishness and not full cooperation can be advantageous and facilitate team learning. In our test environments and with our framework, full cooperation among agents yields superior performance. Depending on the environment, the amount of cooperation and selfishness can be different. Section title: 2 Related work Educational score: 2.883836507797241 Domain: other Document type: Study Language: en The other similar work to ours, which is one of the most popular MARL methods, is the multi-agent deep deterministic policy gradient (MADDPG) that proposed similar frameworks with centralized training and decentralized execution. They tested their method on some Particle environments . Their approach differs from ours in the following ways: (1) They do not have the LSTM (memory) layer in their network, whereas the LSTM layer in the critic network plays a critical role in our method. It helps to learn the interaction and cooperation between agents and also mitigate the partial observability problem. (2) They tested MADDPG on Multi-Agent Particle Environments with discrete action spaces. However, we test our method in both continuous and discrete action space environments. (3) They consider separate critic networks for each agent, which is beneficial for competitive scenarios, whereas we use a single critic network and consider the cooperative tasks. (4) Their method is off-policy with a replay buffer, and they combat the non-stationarity problem by centralized training. In contrast, our approach, in addition to centralized training, is an on-policy method without a replay buffer allowing the networks to use the most recent data from the environment. We will compare our method with MADDPG and show that ours has comparable or superior performance. Wang et al. extends the MADDPG idea and adds a recurrent layer into the networks, but they have separate actors and critics for agents, similar to MADDPG, and recurrent hidden states of critics are isolated, and there is no combination of information in them. They also tested their method on one environment with a discrete action space. Section title: 2 Related work Educational score: 1.1218205690383911 Domain: other Document type: Other Language: en We target problems where agents attempt to collaboratively maximize the sum of all agents’ expected rewards but where each agent receives its reward. We do not specifically consider the credit assignment problem for multi-agent games where all agents have a shared team reward. The proposed algorithm can be applied to such problems, but it is not designed for them. Section title: 2 Related work Educational score: 1.7176467180252075 Domain: other Document type: Other Language: en To provide a clearer comparison, Table 1 summarizes the key differences between MACRPO and other state-of-the-art methods. The table highlights the distinctive features of MACRPO, such as the use of meta-trajectories and a novel advantage estimation mechanism with a cooperation control parameter, which are not present in other approaches. Section title: 3.1 Markov games Educational score: 4.059169769287109 Domain: biomedical Document type: Study Language: en In this work, we consider a multi-agent extension of Partially Observable Markov Decision Processes (MPOMDPs) , also called partially observable Markov games . It can also be modeled as Partially Observable Stochastic Games (POSGs) . A Markov game for N agents is defined by a set of states S describing the possible configurations of all agents, a set of actions U 1 , … , U N and a set of observations O 1 , … , O N for each agent. The probability distribution of the next state as a function of the current state and actions is determined by a Markovian transition function T : S × U 1 × ⋯ × U N → S . Each agent i uses a stochastic policy π θ i : O i × U i → , parametrized by θ i , to choose an action. Upon the state transition, the agent receives a scalar reward r i : S × U i → R . We consider games where the total reward can be decomposed to individual agent rewards r i . Each agent i aims to maximize the rewards for all agents in a cooperative way . Section title: 3.2 Proximal policy optimization Educational score: 3.124285936355591 Domain: other Document type: Other Language: en Proximal Policy Optimization (PPO) is a family of policy gradient methods for solving reinforcement learning problems, which alternate between sampling data through interaction with the environment and optimizing a surrogate objective function using stochastic gradient descent while limiting the deviation from the policy used to collect the data . PPO aims to maximize the clipped expected improvement of the policy ( Equation 1 ). L CLIP θ = E ^ t m i n f t θ A ^ t , c l i p f t θ , 1 − ϵ , 1 + ϵ A ^ t (1) where A ^ t is the advantage obtained by Generalized Advantage Estimation (GAE), ϵ is a hyperparameter, and f t ( θ ) denotes the probability ratio f t ( θ ) ≡ π θ ( u t | o t ) π θ old ( u t | o t ) for importance sampling. The clipping prevents excessively large policy updates. Section title: 3.2 Proximal policy optimization Educational score: 3.5354950428009033 Domain: biomedical Document type: Study Language: en In addition to the expected improvement, the total objective function for PPO incorporates a loss function for a critic network required for GAE and an entropy bonus term to encourage exploration, resulting in the total objective ( Equation 2 ) . L t CLIP+V F+S θ = E ^ t L t CLIP θ − c 1 L t V F θ + c 2 S π θ o t (2) where c 1 , c 2 are weight factors, S denotes the entropy bonus, and L t V F is a squared-error loss for the critic L t V F θ = V θ o t − V t targ 2 (3) In the above equations, V θ ( o t ) is the state-value function and θ denotes the combined parameter vector of actor and critic networks. PPO uses multiple epochs of minibatch updates for each set of sampled interactions. Section title: 4 Methods Educational score: 3.3037803173065186 Domain: biomedical Document type: Study Language: en In this section, we introduce the problem setting and provide an overview of our proposed solution. We then describe the two key components of our approach in detail: (1) a centralized critic based on a recurrent neural network (RNN), which utilizes a novel meta-trajectory to capture inter-agent dynamics, and (2) an advantage estimation technique that incorporates weighted rewards, controlled by a parameter to adjust the level of cooperation between agents. Finally, we present a summary of the proposed MACRPO algorithm. Section title: 4 Methods Educational score: 1.845321774482727 Domain: biomedical Document type: Study Language: en For clarity, a detailed description of all symbols and variables used in the equations throughout the manuscript is provided in the Nomenclature ( Supplementary Appendix A1 ). Section title: 4.1 Problem setting and solution overview Educational score: 1.6179624795913696 Domain: other Document type: Study Language: en Multi-agent systems operating in partially observable environments face significant challenges due to limited information and the absence of direct communication between agents. These factors hinder agents’ ability to coordinate effectively and learn optimal policies, which can lead to suboptimal performance in cooperative tasks. Effective information sharing among agents is critical for improving performance and accelerating learning in multi-agent reinforcement learning (MARL) . In this work, we aim to enhance information sharing in multi-agent environments, going beyond the traditional approach of merely sharing parameters across actor networks. Section title: 4.1 Problem setting and solution overview Educational score: 2.2210121154785156 Domain: other Document type: Study Language: en We introduce the Multi-Agent Cooperative Recurrent Proximal Policy Optimization (MACRPO) algorithm, a cooperative MARL approach based on the centralized training and decentralized execution (CTDE) paradigm. MACRPO addresses the partial observability and lack of direct communication by integrating two novel mechanisms that significantly improve information sharing and cooperation between agents: • Recurrent Critic Architecture : The critic network leverages a recurrent neural network (RNN) trained on a meta-trajectory, which is constructed by combining the trajectories collected from all agents (detailed in Section 4.2 ). This allows the critic to model the interactions and dependencies between agents over time, capturing both agent-specific and collective behavior effectively. • Advantage Function Estimator : A novel advantage function estimation approach that combines the individual agents’ rewards and value functions. This estimator incorporates a control parameter to dynamically adjust the level of cooperation between agents, enabling MACRPO to flexibly handle different cooperation strategies (explained in Section 4.3 ). Section title: 4.1 Problem setting and solution overview Educational score: 1.156567096710205 Domain: other Document type: Other Language: en By combining these two components, MACRPO enables more effective cooperation and improves policy learning in complex, partially observable multi-agent environments, addressing the core challenges of coordination, partial observability, and dynamic cooperation. Section title: 4.2 MACRPO framework Educational score: 1.9710825681686401 Domain: other Document type: Other Language: en The proposed MACRPO framework consists of one recurrent actor, similar to Foerster et al. , and one recurrent critic network, as illustrated in Figure 1 . To consider the partial observability of multi-agent settings, we use recurrent LSTM layers in both actor and critic networks to allow the integration of information over time. Section title: 4.2 MACRPO framework Educational score: 2.0816702842712402 Domain: other Document type: Other Language: en The actor-network architecture is composed of a stack of Embedding, LSTM, and Linear layers and is trained using trajectories collected by all agents. We denote the shared weights of actors with θ a and use the same, latest weights for all agents. The behaviors of different agents vary because of stochasticity and differences in their inputs. Denoting the trajectory data for episode k with length T for agent i as τ i k = o 1 i , u 1 i , r 1 i , … , o T i , u T i , r T i , Section title: 4.2 MACRPO framework Educational score: 1.409597635269165 Domain: other Document type: Other Language: en where o , u , and r represent the observations, actions, and rewards of the agents, respectively. The training data for the actor is then D A = ( τ 1 1 , … , τ i k , … ) . Section title: 4.2 MACRPO framework Educational score: 2.0363142490386963 Domain: other Document type: Other Language: en To enable the critic network to integrate information across both agents and time, we introduce a meta-trajectory , which concatenates the trajectories of all agents during each roll-out. The critic network, consisting of Embedding, LSTM, and Linear layers, is trained on this meta-trajectory, allowing it to capture the interactions between agents and the temporal dynamics of the environment . Section title: 4.2 MACRPO framework Educational score: 2.456705093383789 Domain: other Document type: Other Language: en To ensure that the critic network does not develop a dependency on a specific ordering of agents, we randomize the order of agents each time we generate a meta-trajectory. Specifically, for each meta-trajectory, we fix a random order of agents throughout the trajectory’s time steps, ensuring consistency within the trajectory. However, a different random order of agents is chosen for each new meta-trajectory. This randomization prevents the critic from associating certain positional patterns with specific agents, thereby reducing any positional bias in the learned policy. By varying the order of agents in each meta-trajectory, the critic is encouraged to focus on the agents’ observations, actions, and rewards independently of their positional index, resulting in a more generalized and robust policy that is less sensitive to the order in which agents are presented. Section title: 4.2 MACRPO framework Educational score: 1.9463080167770386 Domain: other Document type: Other Language: en The training data for the critic network is structured similarly to the actor’s training data, but with the meta-trajectory as input. Let the meta-trajectory for episode k of length T for N agents be represented as: μ k = o 1 1 , … , o 1 N , u 1 1 , … , u 1 N , r 1 1 , … , r 1 N , … , o T 1 , … , o T N , u T 1 , … , u T N , r T 1 , … , r T N Section title: 4.2 MACRPO framework Educational score: 1.4650176763534546 Domain: other Document type: Other Language: en The complete training data for the critic is then D C = ( μ 1 , … , μ k , … ) . Section title: 4.2 MACRPO framework Educational score: 2.542133331298828 Domain: other Document type: Other Language: en By leveraging the above meta-trajectory, the critic network receives information from all agents to capture the agents’ history, the interactions between them, and the environment dynamics, all captured by the hidden state. In other words, MACRPO is able to consider temporal dynamics using the LSTM layer, which incorporates a history of states and actions across all agents. Modeling temporal dynamics allows the latent space to model differential quantities such as the rate of change (derivative) between the distance of two agents and integral quantities such as the running average of the distance. Section title: 4.2 MACRPO framework Educational score: 1.3484586477279663 Domain: other Document type: Other Language: en Additionally, the hidden state of recurrent networks can be viewed as a communication channel that allows information to flow between agents to create richer training signals for actors during training. The network will update the hidden state in each time step by getting the previous hidden state and the data from the agent i in that time step. The network architectures for actor and critic are shown in Figures 2 and 3 . It is important to note that the critic network is only needed during training and that the optimized policy can be deployed using only the actor such that the agents are able to operate in a fully distributed manner without communication. Section title: 4.3 Objective function Educational score: 1.5911229848861694 Domain: other Document type: Other Language: en In addition to the LSTM layer, we propose a novel advantage function estimator based on weighted discounted returns using a parameter that controls the agents’ cooperation level and integrates information across agents. We consider the V t targ in Equation 3 as discounted return and propose to calculate it for agent i at time t as Section title: 4.3 Objective function Educational score: 1.856253981590271 Domain: other Document type: Other Language: en Algorithm 1 MACRPO. 1: Randomly initialize actor and critic networks’parameters θ c and θ a 2: for iteration = 1, 2, … do 3: for environment = 1, 2, …, E do 4: Run all N agents with the latest trainedweights in the environment for T time stepsand collect data 5: Combine collected trajectories by allagents according to Figure 1 6: Compute discounted returns and advantageestimates using Equations 4 , 6 7: end for 8: for epoch = 1, …, K do 9: for minibatch = 1, … , M do 10: Calculate the loss functions using Equations 8 , 9 11: Update Actor and Critic parametersvia Adam 12: end for 13: end for 14: end for Section title: 4.3 Objective function Educational score: 3.9563658237457275 Domain: biomedical Document type: Study Language: en R t i = r ¯ t + γ r ¯ t + 1 + ⋯ + γ T − t + 1 V ¯ o T i (4) where r ¯ t = r t i + β ∑ j ≠ i r t j N , V ¯ o T i = V o T i + β ∑ j ≠ i V o T j N (5) where r t i is the reward for agent i at time t , γ is the discount factor, β is the cooperation control parameter used for rewards of other agents, and V ( o T i ) is the value for the final state of agent i . The advantage for each agent i is then calculated as A ^ t i = δ t i + γ λ δ t + 1 i + ⋯ + γ λ T − t + 1 δ T − 1 i (6) where δ t i = 1 N r t i + γ V o t + 1 i − V o t i + β ∑ j ≠ i r t j + γ V o t + 1 j − V o t j (7) where λ is the temporal difference factor of the GAE algorithm, and V ( o t i ) is the state-value at time t for agent i . Section title: 4.3 Objective function Educational score: 3.2579143047332764 Domain: other Document type: Study Language: en The intuition behind the weighting is that each agent’s rewards are likely to be affected most by its own action choice but that the actions taken by other agents can also affect the reward. In addition, the β parameter can be interpreted as a control parameter for the cooperation level between agents, which is manually tuned based on the cooperative nature of the task. This heuristic is related to credit assignment between agents and provides a trade-off between optimizing the policy considering only individual rewards ( β = 0 and no cooperation between agents), which could lead to sub-optimal total reward when individual rewards are in conflict with each other, and optimizing the policy using the sum of all rewards ( β = 1 and full cooperation between agents), which could lead to challenging assignment of credit between agents. One should note that policy optimization is performed across all agents such that in the end, the expected rewards over all agents are maximized, independent of the choice of β . Section title: 4.3 Objective function Educational score: 1.9885960817337036 Domain: other Document type: Other Language: en MACRPO uses separate networks for actors and critics. Therefore, the objective functions of the actor and critic networks are separate, in contrast to PPO. The actor’s objective function in the case of the shared weight is defined as L t CLIP+S θ a = E ^ t L t CLIP θ a + c S π θ a o t (8) and the critic’s objective function is L t V F θ = V θ c o t − V t targ 2 (9) where θ c are the parameters of the critic A parallelized version of the MACRPO algorithm is shown in Algorithm 1 . Section title: 5 Experiments Educational score: 1.7004764080047607 Domain: other Document type: Study Language: en This section presents empirical results to evaluate the performance of our proposed method, MACRPO. We provide a comprehensive evaluation by testing MACRPO across three diverse and well-established multi-agent environments: DeepDrive-Zero , Multi-Walker , and Particle . These environments were selected to represent a range of cooperative multi-agent tasks, differing in terms of action spaces (continuous and discrete), task dynamics, and cooperation requirements, ensuring a thorough assessment of MACRPO’s generalizability. Section title: 5 Experiments Educational score: 1.708147406578064 Domain: other Document type: Study Language: en In addition to evaluating MACRPO, we compare its performance against several state-of-the-art (SOTA) algorithms, including MADDPG , RMAPPO , and QMIX , which are widely regarded as benchmarks in cooperative multi-agent reinforcement learning. The results demonstrate the effectiveness of MACRPO in addressing the challenges posed by these diverse environments. Furthermore, we conduct ablation studies to analyze the impact of each component of our approach, focusing on the meta-trajectory and cooperative advantage function, which are key to improving coordination between agents. Section title: 5 Experiments Educational score: 1.901633381843567 Domain: other Document type: Study Language: en Together, these experiments provide a robust evaluation of MACRPO’s capabilities, demonstrating its adaptability and effectiveness across different settings without the need for additional static experiments. The comprehensive set of results, combined with the comparisons to SOTA methods, illustrates the advantages of our approach in multi-agent reinforcement learning. Section title: 5.1 Test environments Educational score: 2.4933998584747314 Domain: other Document type: Study Language: en We evaluate MACRPO in three benchmark multi-agent environments: DeepDrive-Zero , Multi-Walker , and Particle . These environments were chosen for their diversity in task dynamics, action spaces (continuous and discrete), and cooperation requirements. They provide a comprehensive evaluation of our method’s generalizability across different multi-agent settings.. • DeepDrive-Zero Environment: Several autonomous driving simulators can be used for multi-agent simulation . In this work, we use DeepDrive-Zero , because we do not need to deal with image data and also need a fast simulation environment for training. DeepDrive-Zero is a very fast and 2D simulation environment for self-driving cars that uses a bike model for the cars. We use the unsignalized intersection scenario in this work, which is shown in Figure 4A . To test our algorithm, we consider two cars in the environment, one starts from the south and wants to follow the green waypoints to do an unprotected left-turn, and the other one starts from the north and wants to go to the south and follow the orange waypoints. The agents need to learn to cooperate and negotiate to reach their destination without any collision. • Multi-Walker Environment: The multi-walker environment is a multi-agent continuous control locomotion task introduced in Gupta et al. . The environment contains agents (bipedal walkers) that can actuate the joints in each of their legs and convey objects on top of them. Figure 4B shows a snapshot from the environment. • Cooperative Navigation in Particle Environment: Using the particle environment package from OpenAI , we created a new environment based on the cooperative navigation environment. This new environment consists of N agents and N landmarks, and agents must avoid collisions and cooperate to reach and cover all landmarks. Figure 4C shows the simulation environment. Section title: 5.1 Test environments Educational score: 1.1481188535690308 Domain: other Document type: Other Language: en Each environment presents unique challenges related to agent cooperation, partial observability, and decentralized decision-making, allowing for a thorough evaluation of MACRPO’s performance. Check Supplementary Appendix A2 for more details about the environments. Section title: 5.2 Ablation study Educational score: 3.920511484146118 Domain: biomedical Document type: Study Language: en Four ablations were designed to evaluate each novelty. The name of the method and the explanation shows which ablation has Feed-forward or LSTM or how information is shared in that ablation. In all cases, the parameter sharing proposed in Gupta et al. and Terry et al. was used: • FF-NIC: ( Feed-forward multi-layer perceptron (MLP) network + no information combination ): two feed-forward neural networks for actor and critic. The GAE is calculated using the single-agent PPO GAE equation . There is no LSTM layer or reward and value functions combination for information sharing in this case. • FF-ICA: ( Feed-forward MLP network + information combination using the advantage estimation function ): This case is similar to the previous case, but the GAE is calculated using Equation 6 to show the effect of mixing reward and value functions for information sharing. There is no LSTM layer in this case too. • LSTM-NIC: ( LSTM network + no information combination ): two networks with LSTM layers for actor and critic. There is no information sharing between agents through GAE calculation or the LSTM’s hidden state. The GAE is calculated using the single-agent PPO GAE equation . • LSTM-ICA: ( LSTM network + information combination using the advantage estimation function but not through the LSTM layer ): This case is identical to the previous case, but the GAE is calculated using Equation 6 . • LSTM-ICF: (LSTM network + information sharing using both the advantage estimation function and an LSTM layer in the critic network (full proposed method)) : two networks with LSTM layers for actor and critic. In addition to parameter sharing between actors, the information integration is done through both the advantage estimation function and the LSTM’s hidden state in the centralized critic network, shown in Figure 1 . Section title: 5.2 Ablation study Educational score: 1.685848593711853 Domain: biomedical Document type: Study Language: en Also, in order to see the effect of the β value in Equation 5 and Equation 7 , the proposed method was evaluated with different β values which shows different cooperation levels between agents. Section title: 5.2 Ablation study Educational score: 2.1146063804626465 Domain: biomedical Document type: Study Language: en All experiments were repeated with identical random seeds for each method to reduce the effect of randomness. Hyperparameters used in MACRPO for three environments are detailed in Supplementary Appendix A4 . Section title: 5.2.1 DeepDrive-Zero environment Educational score: 2.363413095474243 Domain: other Document type: Study Language: en We ran all ablations for ten random seeds in the DeepDrive-Zero environment to test our proposed method. We used self-play in simulations and used the latest set of parameters for actors in each episode. The results are shown in Figure 5 . The x -axis shows the number of training iterations. In each iteration, we ran 100 parallel environments for 3,000 steps and collected data. Next, we updated actors and critic networks using the collected data. After each iteration, we ran the agents for 100 episodes, took the mean of these episodes’ rewards (the sum of all agents’ rewards), and plotted them. The shaded area shows one standard deviation of episode rewards. The hyperparameters used in the MACRPO algorithm are listed in Supplementary Table 2 ( Supplementary Appendix A4 ). Section title: 5.2.1 DeepDrive-Zero environment Educational score: 3.7522342205047607 Domain: other Document type: Study Language: en The proposed algorithm, LSTM-ICF, outperforms the ablations. The next best performances are for LSTM-ICA and FF-ICA, which are almost the same. Moreover, information integration in the advantage function, in both FF-ICA and LSTM-ICA, improves the performance compared to FF-NIC and LSTM-NIC; however, the achieved performance gain in the fully connected case is higher. The FF-ICA surpasses LSTM-NIC, which shows the effectiveness of sharing information across agents through the proposed advantage function, even without an LSTM layer. The addition of the LSTM layer in LSTM-ICF further enhances performance by capturing temporal dependencies between agents’ actions and observations, which are crucial in dynamic multi-agent environments like DeepDrive Zero. The LSTM enables the critic to remember past interactions and predict future dependencies, which provides a more holistic understanding of agent dynamics over time. This temporal information allows for more informed decision-making and improves the ability of agents to anticipate and respond to each other’s actions effectively. Consequently, the use of LSTM leads to a more coordinated and adaptive behavior across agents, which is reflected in the superior performance of LSTM-ICF compared to other ablations. Section title: 5.2.1 DeepDrive-Zero environment Educational score: 1.3919280767440796 Domain: other Document type: Other Language: en Figure 6 shows the analysis of the effect of different β values in Equation 4 , Equation 5 , and Equation 7 . The best performance is achieved with β = 1 , which corresponds to full cooperation between agents. In the DeepDrive Zero environment, full cooperation enables agents to make decisions that maximize collective rewards, which is particularly advantageous in tasks that require closely coordinated actions to avoid collisions and optimize traffic flow. Section title: 5.2.1 DeepDrive-Zero environment Educational score: 2.6753814220428467 Domain: other Document type: Other Language: en As β decreases, the level of cooperation between agents is reduced, leading to a decline in performance. Lower values of β (closer to 0) encourage more independent behavior, which can result in suboptimal coordination in this environment, as agents prioritize their own rewards over group performance. This trend illustrates the importance of cooperation in achieving optimal outcomes in tightly coupled tasks, where synchronized actions among agents are critical. Section title: 5.2.1 DeepDrive-Zero environment Educational score: 1.1786986589431763 Domain: other Document type: Other Language: en The β parameter provides MACRPO with flexibility to adapt to different task requirements by adjusting the degree of cooperation. While higher β values are suitable for tasks requiring full cooperation, lower β values may be advantageous in settings where agents have individual objectives but can benefit from limited coordination. While we demonstrate the effect of different β values in this environment, results for other environments are provided only for β ∈ { 0,1 } . Section title: 5.2.1 DeepDrive-Zero environment Educational score: 1.3104511499404907 Domain: other Document type: Other Language: en To achieve smooth driving performance, a curriculum-based learning method and a gradual weight increase of reward factors were used. The weights of Jerk, G-force, steering angle change, acceleration change, and going out of the lane in the reward function were gradually increased to 3.3 × 1 0 − 6 , 0.1, 3, 0.05, and 0.3, respectively. We then added termination of episodes for lane violations to force cars to stay between the lanes. After curriculum learning and smoothing the driving behavior, the cars follow the waypoints to reach their destination. The car that starts from the bottom and wants to make a left turn yields nicely for the other agent if they reach the intersection simultaneously and then make the left turn, and if it has time to cross the intersection before the other agent arrives, it does. A video of the final result can be found in the supplementary materials. Section title: 5.2.2 Multi-walker environment Educational score: 2.3377320766448975 Domain: biomedical Document type: Study Language: en We ran 20 parallel environments and 2,500 time steps during each update iteration for the Multi-Walker environment. After each iteration, we ran agents for 100 episodes and plotted the mean of these episodes’ rewards. Each episode’s reward is the sum of all the agents’ rewards. Ten different random seeds are used for each ablation. We also used the latest set of parameters for all actors. The hyperparameters used in the MACRPO algorithm are listed Supplementary Table 2 ( Supplementary Appendix A4 ). Section title: 5.2.2 Multi-walker environment Educational score: 3.6662240028381348 Domain: biomedical Document type: Study Language: en Figure 7 shows a massive performance improvement of our proposed method, LSTM-ICF with β = 1 , when compared to ablations. LSTM-ICF with β = 0 , which uses information integration through only the LSTM layer, has the next best performance. The LSTM layer enables the critic network to capture temporal dependencies between agents’ actions and states, which is crucial in this environment where agents need to coordinate to balance and move a shared object. By retaining information about past interactions, the LSTM facilitates coordinated behavior, allowing agents to anticipate each other’s movements effectively, which contributes to the superior performance of LSTM-based methods. After these two, LSTM-ICA, which performs information integration using the advantage estimation function, performs better than the FF-ICA, FF-NIC, and LSTM-NIC cases. Section title: 5.2.2 Multi-walker environment Educational score: 3.976304769515991 Domain: biomedical Document type: Study Language: en The effect of the β value and information sharing through the advantage estimation function can be seen in the performance improvement from LSTM-ICF with β = 0 to LSTM-ICF with β = 1 and from FF-NIC to FF-ICA. In the Multi-Walker environment, where agents must coordinate to move a shared object without dropping it, the cooperation level controlled by β plays a critical role. A higher β value (closer to 1) enables agents to prioritize collective rewards, fostering synchronized movement and reducing the likelihood of dropping the object. This emphasis on group performance is reflected in the superior results for LSTM-ICF with β = 1 , where full cooperation between agents leads to optimal performance. In contrast, lower β values (closer to 0) encourage agents to act more independently, which may result in less effective coordination and increased instability in this task. The improvement from FF-ICA to LSTM-ICF further demonstrates the impact of integrating temporal dependencies via the LSTM layer, which is essential in environments like Multi-Walker that require sustained coordination over time. The β value in FF-ICA is set to 1, indicating that it also benefits from full cooperation, but lacks the temporal integration capabilities provided by the LSTM. A video of the trained model can be found in the supplementary materials. Section title: 5.2.3 Cooperative navigation in particle environment Educational score: 2.31545090675354 Domain: biomedical Document type: Study Language: en In the particle environment, in each iteration, we ran 20 parallel environments to collect data for 2,500 time steps and used that data to update the network. The agents were then evaluated using the trained weights for 100 episodes. We ran the simulation with six random seeds. MACRPO hyperparameters are shown in Supplementary Table 2 ( Supplementary Appendix A4 ). Section title: 5.2.3 Cooperative navigation in particle environment Educational score: 2.0768725872039795 Domain: other Document type: Study Language: en The results of this environment are depicted in Figure 8 . Similar to the other two environments, the proposed LSTM-ICF with β = 1 outperforms all ablations. This environment requires agents to coordinate their navigation to avoid collisions, making temporal awareness of others’ trajectories essential. The LSTM layer significantly enhances performance by allowing agents to capture these temporal dependencies, leading to smoother coordination and reduced collision rates. This capability is particularly beneficial in environments with continuous movement and high inter-agent interaction. Section title: 5.2.3 Cooperative navigation in particle environment Educational score: 2.9915802478790283 Domain: other Document type: Study Language: en The effect of the β value is also apparent in the performance differences between LSTM-ICF with β = 1 and LSTM-ICF with β = 0 . Higher cooperation (achieved with β = 1 ) enables agents to prioritize group navigation goals, effectively balancing collision avoidance and path optimization. Lower cooperation levels, as seen with β = 0 , encourage more independent agent behavior, which can lead to suboptimal group coordination in this scenario. The next best performance is achieved with LSTM-ICF with β = 0 , showing that even without full cooperation, the temporal integration from the LSTM layer provides a significant advantage. The LSTM-ICA’s performance is almost identical to LSTM-ICF with β = 0 , indicating that both the LSTM layer and the advantage function contribute similarly to performance improvements, and that information sharing through these mechanisms provides notable benefits over the baseline LSTM-NIC. Section title: 5.2.3 Cooperative navigation in particle environment Educational score: 2.5818493366241455 Domain: biomedical Document type: Study Language: en The results of the ablation study clearly demonstrate the impact of both proposed enhancements—information integration through the advantage function estimation and temporal dependency capture via the LSTM layer—on improving performance. The findings confirm that these improvements are not spurious but are directly attributable to the proposed modifications. Section title: 5.2.3 Cooperative navigation in particle environment Educational score: 2.010892152786255 Domain: other Document type: Other Language: en In general, the cases with the LSTM layer consistently perform better than the feed-forward counterparts, even in the FF-ICA case, which integrates information solely through the advantage function. This highlights the importance of capturing temporal dependencies via the LSTM network for enhanced coordination and navigation in highly interactive environments. Section title: 5.2.3 Cooperative navigation in particle environment Educational score: 2.906083345413208 Domain: other Document type: Study Language: en Our study also explored the role of the cooperation control parameter β , where β = 1 corresponds to full cooperation by optimizing the total reward across all agents, which is the primary objective in these environments. Although values of β less than one were considered as a potential way to mitigate the credit assignment problem, the experiments showed that β = 1 consistently provided the best performance. This suggests that full cooperation is more beneficial in these fully cooperative settings, as lower values of β did not improve credit assignment enough to offset the loss in overall performance. Section title: 5.2.3 Cooperative navigation in particle environment Educational score: 1.5248628854751587 Domain: other Document type: Other Language: en Additionally, while both proposed ideas yield performance improvements, their relative effectiveness varies across environments. In the DeepDrive-Zero environment, information integration through the advantage function has a slightly greater impact than the LSTM layer. In contrast, the LSTM layer is more effective in the Multi-Walker environment, while in the Particle environment, both enhancements contribute equally to performance gains. Section title: 5.2.3 Cooperative navigation in particle environment Educational score: 2.6554577350616455 Domain: other Document type: Study Language: en In our study, the tested environments—DeepDrive Zero, Multi-Walker, and Particle—are fully cooperative multi-agent settings, where agents benefit from high β values that encourage full cooperation. These tightly coupled tasks require agents to work closely to achieve a common goal, making high cooperation levels ideal. However, MACRPO is adaptable and could be applied to partially cooperative environments, where agents have individual goals but can still benefit from limited coordination. In such scenarios, lower β values could allow agents to act more independently while maintaining a degree of cooperation, balancing individual and group objectives. Section title: 5.2.3 Cooperative navigation in particle environment Educational score: 1.3797821998596191 Domain: biomedical Document type: Other Language: en A video of the trained model can be found in the supplementary materials. Section title: 5.3 Comparison to state-of-the-art methods Educational score: 2.2037668228149414 Domain: other Document type: Study Language: en We compared the proposed method with several state-of-the-art algorithms in each environment. Our method is compared against several single-agent baselines with shared parameters across agents (DQN, RDQN, A2C, DDPG, PPO, SAC, TD3, APEX-DQN, APEX-DDPG, and IMPALA), which were tested in Terry et al. . We also compared our method to state-of-the-art multi-agent approaches such as MAGIC, , IC3Net , GA-Comm , CommNet , MADDPG , RMAPPO , and QMIX . Section title: 5.3 Comparison to state-of-the-art methods Educational score: 1.849637746810913 Domain: other Document type: Other Language: en The architecture and hyperparameters used for PPO, IMPALA, A2C, SAC, APEX-DQN, Rainbow-DQN, DQN, APEX-DDPG, DDPG, TD3, and QMIX are taken from Terry et al. which has an open source implementation 1. For MADDPG , we used the original source code 2, for RMAPPO we used their open source code 3, and for MAGIC, IC3Net, CommNet, and GA-Comm we used the open source implementation 4. We performed hyperparameter tuning using grid search to optimize performance for each method. Section title: 5.3 Comparison to state-of-the-art methods Educational score: 1.1362215280532837 Domain: other Document type: Other Language: en Note that some of the official implementations of baselines we used here do not support both discrete and continuous action spaces and we did not modify the code. The non-reported results for some baselines in the paper’s tables and charts are due to this. In addition, we tried to use the discretized version of the DeepDrive-Zero environment for algorithms with discrete action space which may cause poor performance. Section title: 5.3 Comparison to state-of-the-art methods Educational score: 1.2319762706756592 Domain: other Document type: Other Language: en Each agent’s goal in MACRPO is to maximize the total reward of all agents, while the goal of other methods is to maximize the total reward of each agent without considering other agents’ rewards in their objective function. In order to have a more fair comparison, We report the result for our method when β = 0 too. The results are shown in Table 2 . The table contains some empty fields due to the fact that some algorithms do not support continuous or discrete action spaces. Check Supplementary Appendix A3 for more details. Section title: 5.3.1 DeepDrive-Zero environment Educational score: 1.6858128309249878 Domain: other Document type: Study Language: en In this environment, our full method and also the case with β = 0 achieved the highest average reward. The next best was RMAPPO which performed close to our method in the case of β = 0 and then PPO with parameter sharing between agents followed by APEX-DQN and APEX-DDPG. A version of the environment with discretized action space was used for algorithms with discretized action space. Section title: 5.3.2 Multi-walker environment Educational score: 1.4337986707687378 Domain: other Document type: Other Language: en Similar to the previous environment, the proposed method outperformed other methods by a large margin with an average reward of 47.8. Next, PPO with parameter sharing had the second-best performance with a maximum average reward of 41. Our method with β = 0 achieved the third-best average reward. Some algorithms, such as RMAPPO, CommNet, GA-Comm, IC3Net, and MAGIC, do not support continuous action spaces and are marked with a dash in the table. Section title: 5.3.3 Cooperative navigation in particle environment Educational score: 1.4812606573104858 Domain: other Document type: Study Language: en As in both previous environments, our approach outperformed other approaches in this environment as well, although the difference was minor compared to MADDPG. Our method with β = 0 is in the third place after MADDPG with a small margin. We used a categorical distribution instead of a multivariate Gaussian distribution in this environment with discrete action space. Algorithms with continuous action spaces were not tested in this environment, and are marked with a dash in the table. Adapting these algorithms for discrete action environments could be achieved using the same trick, but we did not change the standard implementation for baselines. Section title: 5.3.3 Cooperative navigation in particle environment Educational score: 1.7033703327178955 Domain: other Document type: Study Language: en It is evident from the reported results that RMAPPO performance in the DeepDrive-Zero environment is satisfactory and comparable to our method in the case of β = 0 and that it is average in the Particle environment. As the current implementation of RMAPPO does not support continuous action spaces, we could not test this method in the Multi-Walker environment. Additionally, we conducted hyperparameter searches for RMAPPO, but since this method aims to recommend a set of modifications and hyperparameters that will improve PPO’s performance for multi-agent systems, we did not deviate too far from the main hyperparameters. The performance of MADDPG is not also good in DeepDerive-Zero and Multi-Walker environments. However, it performs well when used in the Particle environment. Section title: 5.3.3 Cooperative navigation in particle environment Educational score: 1.9169503450393677 Domain: biomedical Document type: Study Language: en All hyperparameters for each algorithm are included in Supplementary Appendix A4 . Section title: 5.3.3 Cooperative navigation in particle environment Educational score: 1.4790037870407104 Domain: other Document type: Study Language: en The results show that the performance benefit given by the two proposed ways of sharing information across agents is significant such that the method outperforms state-of-the-art algorithms. Section title: 6 Conclusion and future work Educational score: 2.444455862045288 Domain: other Document type: Study Language: en In this paper, MACRPO, a centralized training and decentralized execution framework for multi-agent cooperative settings was presented. The framework is applicable to both discrete and continuous action spaces. In addition to parameter sharing across agents, this framework integrates information across agents and time in two novel ways: network architecture and the advantage estimation function. An ablation study in three environments revealed that both ways of information sharing are beneficial. Furthermore, the method was compared to state-of-the-art multi-agent algorithms such as MAGIC, IC3Net, CommNet, GA-Comm, QMIX, and MADDPG, as well as single-agent algorithms that share parameters between agents, such as IMPALA and APEX. The results showed that the proposed algorithm performed significantly better than state-of-the-art algorithms. Section title: 6 Conclusion and future work Educational score: 1.1949845552444458 Domain: other Document type: Other Language: en While MACRPO demonstrates strong performance in a range of tasks, there are some limitations. In environments with high-dimensional discrete action spaces, performance may be impacted due to the increased complexity of managing agent interactions. Additionally, MACRPO’s centralized critic, which processes a meta-trajectory of all agents, may face scalability challenges as the number of agents grows. For larger agent populations, integrating an attention mechanism could be a potential solution, allowing agents to selectively focus on critical information from others without processing data from all agents simultaneously. This enhancement could improve MACRPO’s efficiency and scalability in large-scale multi-agent environments. Section title: 6 Conclusion and future work Educational score: 1.1386140584945679 Domain: other Document type: Other Language: en Despite these limitations, MACRPO’s adaptable cooperation control parameter, β , makes it highly flexible for diverse multi-agent tasks, such as autonomous driving and collaborative robotics, where varying levels of cooperation are essential. Future work could further explore adaptive cooperation strategies and attention-based architectures to enhance MACRPO’s application scope and performance in complex, large-scale multi-agent systems. | Other | other | en | 0.999997 |
PMC11695848 | Section title: Introduction Educational score: 1.0476090908050537 Domain: other Document type: Other Language: en The technological advance of the last 10 years has relentlessly offered us new possibilities to interact with each other and, as in any modern society, communication through the Internet or digital means has become the predominant way of interaction that minimizes the effort. Technologies are developing more and more rapidly and are changing the way we operate in modern society. In this regard, new technologies are continuously improving, bringing more and more opportunities to simplify interactions between individuals. Although this rapid advance of new communication technologies brings many advantages, these digital means also generate a series of spaces and environments conducive to the propagation of unwanted behaviours in the sphere where we also frame the phenomenon of Cyberbullying. Next, it represents a digital form of harassment with significant differences in response, personality, form and content. At the level of recent years, the phenomenon of Cyberbullying, due to the prevalence of this ample phenomenon, has become the subject of international interest. Section title: Introduction Educational score: 1.198718547821045 Domain: other Document type: Study Language: en The study has the main role of analyzing and studying the phenomenological aspects related to cyberbullying or the so-called phenomenon of “cyberbullying” and aims to bring more knowledge regarding the way of spreading this phenomenon in the virtual environment, trying to explain the triggering causes and identify the main solutions both to combat and to prevent it by analyzing all at once the history of its occurrence and propagation in what we call social media today. In this regard, the purpose of our paper was to analyze the way the phenomenon of cyberbullying manifests in terms of frequency on social media platforms, following all at once the nature of gender factors as well as the analysis of elements related to the behaviour/reactions of witnesses and victims. Considering the objectives of the research, in our study we explored the role of witnesses and victims in the experience of cyberbullying on social media, we looked at the main forms of cyberbullying encountered by students on social media, we looked at the degree of awareness that students have about this phenomenon, and we also looked at the frequency with which students use social media and if their opinions and experiences differ depending on gender. Section title: Introduction Educational score: 1.5594173669815063 Domain: other Document type: Study Language: en In the elaboration of this study, we used the scientific method as a basis for acquiring new knowledge based on tangible evidence. In order to conduct the research, we used quantitative methods, the research instrument being represented by a questionnaire. The answers obtained were subsequently systematized in a database, processed and interpreted using the IBM SPSS version 23 statistical processor. Looking at the novelty element, the current study offers an updated vision regarding the study of the phenomenon of bullying on the Internet in Romania compared to the level of this phenomenon in 2022. In addition, the current study could also be developed and extended by studying the phenomenon of cyberbullying at international level. In our study we took into account the perspective of students from Romania regarding their experiences with cyberbullying and regarding their perception about this phenomenon. Thus, while taking into account the view of a similar study, which argued that in order to try to prevent bullying, is important to “reach a consensus among children and adults” regarding the forms and meaning of cyberbullying , through our study we highlight the importance of analyzing cyberbullying from the perspective of young people. In other words, by examining the perception of students about cyberbullying we can have a more clear view on how they perceive and understand this phenomenon. Hence, this perspective is important at the level of Romania, because cyberbullying has been poorly studied at the level of our country, and it can be important at an international level, because it can offer insights into the way students perceive and experience cyberbullying. Moreover, the results obtained through this study, could be later compared to the results of similar international studies which approach cyberbullying from the perspective of students. Section title: Introduction Educational score: 1.8270066976547241 Domain: other Document type: Study Language: en In the first phase, the current research seeks to bring an overview of the issues studied and we sought to achieve a clear, conceptual delimitation between the traditional form of bullying and cyberbullying, highlighting the specific features and forms of each concept, thus reaching the idea that these two forms of abuse are not exactly different by only taking into account the space in which they manifest themselves but also by considering the outline they have, and the component characteristics they incorporate. Hence, even though sometimes, cyberbullying is regarded as simply a type of bullying that takes place in the online environment, we argue that cyberbullying has some characteristics that are different from the characteristics of face to face bullying. In line with this view, a previous study , argued that in the case of cyberbullying, there is no physical contact between people and usually the identity of the aggressor is unknown. In other words, one of the characteristics that differentiates face to face bullying from cyberbullying is the fact that the aggressor is anonymous. Furthermore, even though to some degree, cyberbullying can manifest online in the same forms it can also manifest face to face, such as gossiping, spreading rumors or humiliating other people, a previous study emphasized the fact in the case of cyberbullying, due to the characteristics of the medium in which it takes places, it can be very difficult to realize and identify the identity of the perpetrators . Thus, the Cyberbullying phenomenon refers to the repeated and intentional behaviour of injury exercised mainly in the online space and it represents a real problem of the modern world focused on advanced communication technologies. This phenomenon manifests itself among adolescents and young people, especially because of the many possibilities in the cyber area. In this regard, our study seeks to expose the predominant propagation mode, to analyze and detail general aspects related to the depth its propagation through social networks (social media) that present the main consequences of a psychosocial and general nature as a negative consequence of this phenomenon. In the context of cyberbullying among university students, the development of technology can facilitate the phenomenon of cyberbullying. Thus, students access and use frequently a wide range of social networks which offer them many option of interacting with one another, such as: direct/private messages, public messages posted on the accounts of other users, or messages sent as a response to certain posts. Moreover, the use of social networks for academic purposes, like having certain online groups for studying and sharing ideas about the projects developed, can extend bullying in the context of social/personal interactions to bullying in the context of the educational process. Hence, the analysis of the cyberbullying phenomenon on social media is relevant because social networks can facilitate the process of bullying in a number of ways. Compared to face to face bullying, on social networks perpetrators can keep their anonymity, the act of shaming people can be done in front of a large audience, but social networks could also help people report the cases of cyberbullying that they have witnessed or experienced. Section title: Purpose and objectives of the research Educational score: 1.125738501548767 Domain: other Document type: Study Language: en The current study aims to analyze the way the phenomenon of cyberbullying manifests in terms of frequency on social media platforms, following all at once the nature of gender factors as well as the analysis of elements related to the behaviour/reactions of witnesses and victims. Thus, the specific objectives of the research approach are: Section title: Hypotheses of the research Educational score: 1.7447758913040161 Domain: biomedical Document type: Study Language: en Related to the research conducted we formulated the following research hypotheses: Section title: Clarifying the concept of cyberbullying Educational score: 0.9953252673149109 Domain: other Document type: Other Language: en The development of social networks and the advance of GSM (Global System for Mobile Communication) technologies have become natural things in the contemporary society. The new generations of people benefit from quick access to information and are becoming more and more attracted to virtual environments where well-known social media platforms are also found. The development of such platforms has facilitated the construction of a virtual space where individuals are free to create and upload photo-video content, relate and even carry out online orders and purchases. In addition to these main advantages, these platforms can also generate unpleasant experiences where we identify a recent phenomenon, namely, Cyberbullying. “Unlike traditional bullying, cyberbullying is mainly differentiated by form and medium of manifestation” and due to the novelty of the phenomenon we encounter a certain lack of specialized materials regarding the concept behind the term. From an etymological point of view, the term “cyberbullying” is defined as “knowingly harming that has a repetitive character and has the main starting point of text messages” . From another perspective, the term cyberbullying is also defined as “an act of violence that is well-oriented by an individual or group of individuals using electronic technologies against a defenseless person” . Thus, considering the diversity of the ways in which cyberbullying is defined, a previous study describes cyberbullying as a form of using digital tools in order to directly or indirectly target people, and while describing the phenomenon, the study emphasizes the fact that one should take into account the perspective of the perpetrator, the perspective of the victim as well as the perspective of the bystander . Often, the term Cyberbullying is related to other terms and explained as a type of abusive behaviour, abuse or harassment. According to other sources, abusive conduct is explained as a “type of behaviour that is intended to injure the victim by causing psychological harm” . Section title: Clarifying the concept of cyberbullying Educational score: 1.0662888288497925 Domain: other Document type: Other Language: en As for harassment, this is a form of abuse that consists of a “series of actions that are meant to negatively affect the individual or their group of belonging” . From another author’s perspective, the American term “cyberbullying” is also defined as a “violent behavioural type generated through devices by aggressors who seek to cause harm to an individual or group” . Currently, cyberbullying has been defined as “harmful and intentional behaviour that is carried out by an individual or group or individuals, which has a repetitive character, using modern digital technology to assault a victim who is incapable of defense” . The aggressor is stronger in some respects than the target and this definition works in parallel with the definition of traditional harassment, essentially adding the concept of “digital technology” as the main mechanism by which the harm to the victim is caused. In addition to all these main features of the phenomenon (intentionality, repetitiveness, power imbalance), several researchers have suggested that “anonymity is an additional characteristic that defines the phenomenon of cyberbullying” . In the context of the anonymity theory, in a general manner, anonymity can be described as the state of not being able to be identified, or as the state of being unknown . In this regard, anonymity may contribute to the development of the phenomenon of cyberbullying, because in the online environment, the aggressor can remain anonymous, there is no need for the aggressor to know or to have previous interactions with the victim, and the victim does not possess any physical evidence, such as scars, from the process of bullying . Moreover, previous studies on the matter of anonymity and cyberbullying , highlighted the fact that in the online environment people feel that they have more anonymity and this perceived anonymity could facilitate the process of cyberbullying. Section title: Clarifying the concept of cyberbullying Educational score: 1.063172698020935 Domain: other Document type: Other Language: en The overall characteristics of the digital environment can significantly increase the risks of cyberbullying. These are represented by: the size of the potential audience; continuous access to information; the permanence of online content; the ease of copying the material and distributing it widely and the lack of oversight of behaviors on social media platforms. Section title: The main forms of the cyberbullying phenomenon Educational score: 0.9308288097381592 Domain: other Document type: Other Language: en The presence of individuals in the online environment increases significantly from year to year. Daily, people use gadgets such as mobile devices and other sources of communication and entertainment to facilitate communication and often they turn to social media platforms and text messages without taking into account the possibility of exposure to less obvious risks. Because this alarming phenomenon has spread rapidly in the online environment, specialized studies have stated that cyberbullying manifests itself in a wide range of ways and these must be analyzed and tracked differently depending on its specific implications. A main pioneer in the research of this ample phenomenon has identified and catalogued a wide range of forms specific to this phenomenon of “where we count: Denigration; Exclusion; Harassment; Outing; Inflammation (flaming); Online tracking (Cyberstalking); Impersonation of False Identities (Impersonation) and Deception (Trickery)” . Section title: The main forms of the cyberbullying phenomenon Educational score: 1.006388783454895 Domain: other Document type: Other Language: en At the moment, there are numerous forms of manifestation of the Cyberbullying phenomenon and most often, it is noted that these forms are combined by the aggressor to maximize the efficiency of obtaining what he/she originally set out to do. The combination of methods varies depending on the object pursued by the aggressor but also on the typology and characteristics of the victim or group on which this ingratiated form of abuse is committed. To date, there is no specific institution meant to combat and control this form of abuse, and this is shown by the increased rate of Internet users who have been victims of cyberbullying in the last year at international level. Section title: The concept of social media Educational score: 1.0671186447143555 Domain: other Document type: Other Language: en Today’s social media platforms and applications allow individuals to connect and interact in an easy way and such platforms made possible the creation and development of large virtual communities, something that until the advent of Web 2.0 was not possible. Section title: The concept of social media Educational score: 0.9048097133636475 Domain: other Document type: Other Language: en Currently, perhaps the handiest definition is the one that describes the social media space as “those internet-based interpersonal communication channels that facilitate mass personal communication and encourage interaction between users” , the term “social media” being used for the first time in 1994 and during the development of the Internet the first social media platforms were conceived and launched. Over time, both the number of social media platforms and the number of active users have increased significantly, thus making them some of the most important and attractive parts of the Internet that we know today. “Social media” is used in general as a term that describes a variety of online platforms including blogs, social networks, forums, microblogs, and video photo content-sharing networks. Given this wide spectrum of social media platforms, social media applications are also quite diverse and are not limited to instant content sharing. As of January 2021, there are over 130,000 publications that have the term “social media” included in the title. In articles published over the past 20 years, several authors have formulated several quite different definitions of the term “social media” sometimes using alternative terms. Reporting to the level of publications from the last 12 years to the present, the term social media is beginning to be defined in a variety of ways updated and reported in the context of the continuous development of social media platforms and websites. Section title: The concept of social media Educational score: 1.0360701084136963 Domain: other Document type: Other Language: en In the beginning, in the early development of the Internet and social networks at the level of 2010, the term “social media” was defined simply by referring to “that group of internet-dependent applications that are based on web 2.0 technology and that allow the creation, exchange and distribution of user-generated content” . A year later, in 2011, a more theoretical definition is presented which exhibits several characteristics. According to her, the term “social media” refers to “a construct consisting of seven great characteristics: identity, conversation, sharing, presence, relationship, reputation and group” . Section title: The concept of social media Educational score: 0.9063853621482849 Domain: other Document type: Other Language: en Analyzing the last years and taking into account the substantiation of the social media space already in its period of maturity, several authors have found and developed four important definitions that are the closest to the social media space that we know today. Thus, the term and concept of “social media” received updated definitions that are related to the continuous advance and development of recent years, where we list: From the perspective of Carr and Hayes , social media “represents those mass communication channels based on internet connection that facilitate connections and interactions between users generating added value through user-generated content.” From the perspective of Miller et al. the social media environment is “the space between traditional communication and private dyadic communication that gives individuals several degrees of intimacy.” From the perspective of Leyrer-Jackson and Wilson , social media refers to “those websites and digital applications that allow its users to share content…” From the perspective of Kapoor et al. the social media environment refers to “those networks that encompass various user-led platforms that facilitate the dissemination of content, the creation of dialogue and communication to the general public.” In essence, this is a digital space created by the individual, for the individual and provides an environment conducive to the development of interactions and the creation of personal and/or professional bonds. Thus, looking at the multitude of definitions and approaches, we argue that social media currently refers to any resource conceived and developed to facilitate a mediated connection between individuals or social actors. Section title: Cyberbullying and social media Educational score: 1.040579915046692 Domain: other Document type: Other Language: en Nowadays, there are various ways in which aggressors harass their victims. From the traditional (physical) harassment that manifests itself in the neutral spaces of the aggressor and the victim, the technological advance generated by the development of the social media environment, that includes various applications and social networks, has allowed “traditional” harassment to take on a new form through its implications, namely the form of cyberbullying. Cyberbullying means “the intentional act of intimidation used by the aggressor against the victim through social media” , and this seemingly out-of-control phenomenon involves “threatening, denigrating, stealing information and content, blackmailing and spreading false rumors in direct relation to the victim targeted by that cyber aggressor” . Section title: Cyberbullying and social media Educational score: 1.0409139394760132 Domain: other Document type: Other Language: en The negative effects of cyberbullying are numerous, and the consequences of cyberbullying “involve poor professional and personal performance, decreased motivation, physical violence, and suicidal behaviours that are often hidden or masked by the victim” . From the perspective of Patchin and Hinduja , the phenomenon of cyberbullying is closely related to a series of effects that generate clear negative consequences such as low self-esteem, family problems, academic problems, violence and delinquent behaviour, and yet the most serious consequence is represented by the suicidal behaviour of the victim. While cyberbullying generates some of the riskiest negative effects as in the case of traditional (face-to-face) bullying, “cyberbullying harassment manifests itself in the absence of physical contact and often without knowing the identity of the perpetrator” . Section title: Cyberbullying and social media Educational score: 0.9689522385597229 Domain: other Document type: Other Language: en These random acts of cyberbullying go far beyond the scope of the action in terms of traditional (face-to-face) harassment because, unlike traditional harassment, “cyberbullying can manifest itself not only in the common spaces frequented by the aggressor and the victim but also in the personal space or in any other place where the technology is available to the average user and thus accessible” . Further, several studies started in this regard have suggested that although cyberbullying may occur and manifest less frequently than traditional (face-to-face) harassment, up to 70% of the total number of respondents to these studies conducted in the United States have stated that they have faced several behaviours in recent years that have been based on cyberbullying . Section title: Cyberbullying and social media Educational score: 1.0180953741073608 Domain: other Document type: Study Language: en With the development of new communication technologies through social networks and applications, the phenomenon of cyberbullying has spread and rapidly propagated in this environment since the appearance of the first social network in 1997–1998: SixDegrees, for the simple reason that users predominantly use such platforms that have led to a paradigm shift in the way we interact daily. Thus, apart from facilitating communication and interaction between people, social networks have also provided a dangerous environment where, not infrequently, users have become the targets of cyber aggression. In this regard, bullying through social networks is “a form of manifestation of aggressive behaviour coordinated by an individual or, as the case may be, a group of individuals who exercise a negative behaviour repeated over time and which is directed against often defenseless people” . This type of bullying is clearly distinguished from other forms of deviant behaviour by the simple fact that it is intentional and directed by the aggressor to a safe victim using new social media communication technologies. Often, this form of deviant behaviour was made possible “by the emergence and development of new information technologies that led individuals to spend more and more time on the internet and implicitly on social media sites and networks” . Hence, in the context of social networks, while studying the phenomenon of cyberbullying on TikTok, a previous study showed that TikTok creaters often received hate comments on their videos, and most people received comments from other people who are protecting their identity by having profile pictures that do not represent them. Furthermore, in the context of cyberbullying and Snapchat, a previous study focused on analyzing the perception of educators and practitioners in schools, about the use of Snapchat among students and the study emphasized that due to the features of the platform, cyberbullying and acts of harassment could be magnified on this platform . Section title: Cyberbullying and social media Educational score: 0.9540454745292664 Domain: other Document type: Other Language: en The change in social activities and the transition from the offline environment to the social media platforms has led to the creation of new possibilities for aggressors to track and harass victims and this was clearly because the large number of social media users has automatically generated the creation of numerous profiles through which the aggressors can more quickly identify their victims. In terms of combating these acts of harassment, it is impossible to eradicate this phenomenon as millions of daily social networks interactions are present, and so it is very difficult to monitor and control all interactions that have violated community policies and standards. “This perspective is thus consistent with the theory of the expediency of crime, which states that social change generates new opportunities in terms of crime and deviant behaviour” . The theory of the expediency of crime states that “social and technological changes relentlessly produce new opportunities for crime and deviant behaviour” . Opportunities in this way play a central role regardless of the nature or severity of the crime and this perspective suggests that changes in social activities (moving from offline to online) offer aggressors new possibilities in terms of engaging in actions specific to cyberbullying. Section title: Cyberbullying and social media Educational score: 1.0405831336975098 Domain: other Document type: Other Language: en The increase in the number of users creates the right environment for the manifestation of cyberbullying behaviours from the simple fact that aggressors can identify vulnerable people with some ease by viewing the public profiles present on social networks and social media applications . Furthermore, in the context of the forms that cyberbullying can take depending on the type of social network used, a previous study showed that Facebook was the platform on which users mostly experienced cyberbullying, in the form of posting mean comments or spreading rumors . On Instagram, for example, due to its visual nature, cyberbullying can happen by posting inappropriate images of someone, but similar to the case of Facebook, it can happen through hateful comments, harassment or denigration . Section title: Materials and methods Educational score: 3.6683390140533447 Domain: biomedical Document type: Study Language: en In order to conduct the research, the scientific method was used, which thus represents the typical way in which “science continues to reach an objective, reliable, verifiable and shareable knowledge of reality thus consisting in the collection of empirical data under the guidance of working hypotheses and theories existing and present to empirical knowledge” . Regarding the research methodology approached, our research is descriptive, therefore, what we intended to achieve was to define, classify, divide and summarize the problem studied through this type of research. Further, according to the methodology of the proposed research, this study takes the form of quantitative research because it aims to generate models, hypotheses and work theories. Thus, in this descriptive research we used the survey method which represents “an indirect method that is used mainly for the determination of personality traits, attitudes or mentalities that cannot be “brought” to the laboratory” . Thus, we further substantiated the research approach using the survey technique while having as an instrument a questionnaire, which is an important component of the system of methods in the social sciences. Section title: Sample Educational score: 1.8356482982635498 Domain: other Document type: Study Language: en The population from which we selected the respondents consists of all the students of Transylvania University of Brasov, a total number of 20,284 students according to the last annual report of the university at the level of the year 2022, from all 18 faculties and all the specializations and educational programs (Bachelor, Master, PhD), that are present and active on the Facebook group created by the students: “Univ. Transilvania din Brașov.” As far as sampling is concerned, this represents “the strict selection, from among several sources of information, of the class of documents that is most relevant to the topic of study and the research objectives” . In this research, we used the simple random sampling meth od (non-probabilistic) or the so-called randomized sampling which is a simple method by which the subjects (individuals) of the investigated population are randomly selected and thus, each individual has the same chance to be selected and included as the respondent of the study. Section title: Sample Educational score: 1.6841145753860474 Domain: other Document type: Study Language: en The research was conducted between March and April 2023, and the volume of the research is represented by 500 respondents: students of Transylvania University of Brasov present and active on the Facebook group where the research tool, the questionnaire, was shared and applied. The respondents included in the study have the following characteristics: 50% were males and 50% were females; 78% of them are undergraduate students and 22% master students; 59% of respondents come from urban living areas and 41% come from rural living areas; 54% of them are students with the age between 18–21 years; 33% with the age between 22–25 years and 13% with the age between 26–30 years; 95% of respondents stated that they hold Romanian citizenship. Section title: Sample Educational score: 2.194516181945801 Domain: biomedical Document type: Study Language: en The minimum sample size was tested with the G*Power 3.1.9.7 software and was detailed in each hypotheses proposed to be tested below. The interpretation of the results followed the suggestions of a previous study . For this research we conclude that our sample of 500 respondents was large enough to exceed the minimum size threshold in each case. Section title: The research tool Educational score: 1.6998671293258667 Domain: other Document type: Other Language: en The research tool used in order to conduct the research is the questionnaire. According to the classification of Chelcea , depending on the content of the information obtained, the tool used is an omnibus questionnaire that addresses several research topics/ objectives and represents the most common type of questionnaire in sociological research. Next, this type of questionnaire is characterized by a large amount of information on social processes and can also encompass and capture interactions or inter-conditioning between them. Analyzing another criterion, namely the type of questions used, this research tool includes a series of pre-coded (closed) questions to which the variants of answer are pre-established and the respondent thus follows the choice of a variant of answer by his/ her opinion. Section title: The research tool Educational score: 1.7462140321731567 Domain: other Document type: Study Language: en In addition to this type of questions, in the construction of the questionnaire, we have also introduced a series of open-ended questions to which the answers are not established in advance, to offer to the respondents the opportunity to freely express their opinions. Finally, the third classification criterion proposed by Chelcea concerns how the questionnaire is applied and in the present case, this is a self-administered questionnaire because the respondents included in the sample answered to the questions by accessing the questionnaire that was posted in the online environment on the Facebook group of our university. Regarding its elaboration, the questionnaire was transcribed/ typed on the Google Forms platform and the responses obtained were exported in the form of an Excel document and imported into the Statistical Package for Social Sciences (SPSS), version 23, made available by International Business Machines Corporation (IBM). The questionnaire can be found in Appendix A . Section title: The research tool Educational score: 3.4257712364196777 Domain: other Document type: Study Language: en Given the analysis of the data collected, we used some specific statistical methods, such as chi-square and factor analysis, because these were the appropriate methods which helped us test the hypotheses of the research and implicitly to see if they are confirmed or not. Hence, in our analysis, we included as predictors gender, types of studies and age categories. To test the hypotheses, we used: the Chi-Square Test of Independence (for Hypothesis H1 and H3), a non-parametric Mann–Whitney U test and nonparametric Kruskal-Wallis Test (Hypothesis H2). For the hypothesis H2 we applied a Principal Components Factor Analysis. The results delivered a single factor (with eigenvalue = 3.29 > 1) that explained 41.23% of the variance in responses. With this analysis we defined our statistical cumulative index named ‘bullying index’. Considering the structure of the questionnaire, it includes a total of 24 questions arranged as follows: the questions from the number 21 to 24 are sociodemographic questions, meant to identify the respondents and to describe the respondents through aspects and information related to gender, age, citizenship, environment of residence and level of education. These questions have a number of variants of answer and take the form of closed pre-coded and open-ended questions. The establishment of the frequency values from question 11 to 18 represents the encoding in variables of the 8 dimensions of the Cyberbullying phenomenon and represents items / values measurable on the Likert scale. Question number 10 is a question that encompasses the scale of social distance that takes into account the measurement of social relations between the actors involved in this phenomenon. Question 1 is a closed pre-coded question that has the role of determining the level / awareness of the phenomenon on social media. Question 2 is a question that takes into account the measurement on the Likert scale of the frequency of use of social media by the respondents involved in this study. Question 3 is intended to determine whether the respondent has been a victim of the Cyberbullying phenomenon. Question 4 is related to question 3 and is intended to go into detail on the platform on which the event happened. Question 5 is an opinion question regarding the respondent’s considerations about social media platforms and the pre-availability of the phenomenon studied on them. Towards the end, the questions from number 6 to number 9 are aimed at establishing the main behavioural reactions of the respondents both as victims and as witnesses of the cyberbullying phenomenon. Section title: The research tool Educational score: 1.0217586755752563 Domain: other Document type: Other Language: en To summarize the dimensions mentioned through the questions included in the questionnaire, question 1 measures the dimension regarding students’ awareness of the phenomenon on social media, question 2 measures students’ frequency of use of social media. Question 3 refers to the dimensions referring to whether the respondent has been a victim of cyberbullying and question 4 measures the dimension of the platform on which cyberbullying happened. Question 5 measures the dimension of the students’ opinion about the prevalence of cyberbullying on social media platforms. Questions 6 to 10 measure dimensions related to the main behavioural reactions of the respondents both as victims and as witnesses cyberbullying. Questions 11 to 18 measure the dimension referring to forms of cyberbullying. Question 19 measures the dimension referring to a relation between offline and online bullying, and question 20 measures the dimension related to students’ opinion about the ways cyberbullying can be combated. Questions 21 to 24 measure sociodemographic dimensions. Section title: The research tool Educational score: 1.116472840309143 Domain: other Document type: Study Language: en Given the Likert scale used in the context of measuring the forms of cyberbullying, we used a 5 point Likert scale for frequency: 1—never, 2—rare, 3—several times, 4—frequently, 5—very frequently. The scale measuring the perceived degree of bullying has good reliability (Alpha Cronbach = 0.709, number of items = 8). A frequency scale was considered appropriate because the respondents were asked how frequently they have been a victim in a verbal conflict, how frequently they have been harassed or gossiped about on social media, how frequently someone pretended to take their identity online, how frequently they were excluded from social media groups or they have been threatened on social media. In this regard, the Likert scale used helped us identify how often students experienced various forms of cyberbullying. Section title: The research tool Educational score: 1.0345990657806396 Domain: other Document type: Other Language: en Furthermore, considering the way the questionnaire was designed in order to measure the experiences of witnesses and victims, we firstly included in the questionnaire a filter questions through which we asked the respondents if they have been victims of cyberbullying. Then, for those who responded positively to this question, we included an additional question referring to the platform on which the cyberbullying took place. Next, the matter of students being victims of cyberbullying was measured through questions 11 to 18, which were supposed to reveal how often students experienced different types of cyberbullying. Then, for the people who were not victims of cyberbullying, we designed questions referring to the types of cyberbullying they have witnessed or seen in the case of other people on social media, questions referring to the way they reacted when they witnessed cyberbullying, to the reasons why people resort to cyberbullying on social media, to the way they would react if they would be victims, or to the people they would speak to if they would experience cyberbullying (questions 6 to 10). Section title: The research tool Educational score: 1.643912434577942 Domain: other Document type: Study Language: en Hence, the questionnaire was pre-tested on a number of 50 students from Transilvania University from Brasov, in order to assure the clear understanding of the open-ended questions and of the close-ended questions. After the pre-testing process, the students confirmed that they did not encounter any difficulties in understanding and answering the questions. Section title: The research tool Educational score: 0.9963447451591492 Domain: other Document type: Other Language: en Moreover, taking into account the objectives of our research and the connection between them and questions included in the questionnaire, in the context of the first objective: to establish the main form of cyberbullying that the investigated population has most often faced on social media, questions 11 to 18, refer to the main forms of cyberbullying that the respondents faced on social media. In the context of the second objective: to determine which of the genres was more exposed to the phenomenon of cyberbullying, question 21 regarding the gender of the respondents is relevant, as well as questions 3 and 4. In the context of the third objective: to establish to what extent the investigated population is aware of the presence of the phenomenon on social media networks, question 1 is relevant and in the context of the fourth objective: to determine how often the investigated population uses social media platforms, question 2 is relevant. Section title: The research tool Educational score: 1.8506358861923218 Domain: biomedical Document type: Study Language: en For the questionnaire validity and reliability, we follow the suggestions of Fowler . To increase the validity of the questionnaire we documented other research with very close subject and we pre-test our questions with specialists form our departments, other colleagues from other universities. We summarized all the suggestions to estimate to what extent our work tool measures what it set out to measure. For reliability we applied the questionnaire on a random sample of two group of students and we repeat this research after three mounts. We found that there were no significant statistical differences in the test–retest reliability. Section title: Results Educational score: 1.075761318206787 Domain: other Document type: Other Language: en From the beginning, respondents were asked to specify how often they use social media platforms like Facebook, Instagram, Snapchat, etc. Table 1 presents the frequency with which the respondents use social networks. Section title: Results Educational score: 1.0953609943389893 Domain: other Document type: Other Language: en We can observe from Table 1 that 75% percent of students declared that they used social networks often and very often. The percentage of respondents who do not use social networks is very low. Section title: Results Educational score: 1.128433108329773 Domain: other Document type: Study Language: en Another point of interest was the extent to which the respondents are aware of the presence of bullying on the Internet, with a high incidence, first of all, on social communication networks. Not surprisingly, 96% of the respondents stated that the phenomenon exists on social networks (a fact recognized not necessarily from direct experiences). This unanimity confirms that bullying is no longer an unknown phenomenon, it is easy to identify and, at least theoretically, it should be easy to counteract. In this regard, we tested the first hypothesis of our research: Section title: Results Educational score: 3.9229159355163574 Domain: biomedical Document type: Study Language: en To begin with, we used the G*Power software to calculate the minimum sample volume in the case of applying the Chi Square test of independence. In our case, for effect size ω = 0.3, alpha = 0.05 and df = 2, we obtained a minimum sample size of 172 subjects. This sample represents the minimum number of observations to be able to assume the statistical effect of type I error probability and power. Section title: Results Educational score: 1.8593578338623047 Domain: biomedical Document type: Study Language: en The results obtained from testing the hypothesis are further presented in Table 2 . Section title: Results Educational score: 1.7402291297912598 Domain: other Document type: Study Language: en To test hypothesis 1, we analyzed the association between the age variable and the dichotomous variable regarding the fact of being harassed on the Internet. Starting from the obtained sample, we grouped the respondents into three age categories: 18–21 years old (with reference to students from the first and second year of studies who are at the beginning of their university education), age 22–25 years old (with reference to students finishing their undergraduate studies) continuing with students between the ages of 26 and 30 (referring to students generally pursuing master’s studies). This division of the sample by age was used to see to what extent the three categories of respondents are significantly different in terms of exposure to online bullying. Considering the results from Table 2 , we note that the association of the two variables was statistically significant ( χ 2 (2) = 60.949, p = 0.000). Thus, we can conclude that younger students are more exposed to bullying on social media. Section title: Results Educational score: 1.288040041923523 Domain: other Document type: Study Language: en Moreover, if we consider 100% of those who declare themselves harassed on social media and distribute them by age category, we noticed that 64.9% were from those aged 18–25, 31.6% were from the category of those aged 22–25 years old and only 3.5% belonged to the third category, i.e., 26–30 years old. The magnitude of the effect had the value phi = 0.349 (p = 0.000), so a value justifying an association of moderate intensity but statistically significant. The first hypothesis of our research was confirmed. Hence, younger students could be more exposed to bullying on social media due to reasons related to their perception about the content they see online, their perception about the way people talk online with them. In other words, it is possible for younger students to be more sensitive to criticism ore negative feedback, or to interpret some messages as being directed towards them in a negative manner. Section title: Results Educational score: 0.9901502728462219 Domain: other Document type: Other Language: en Accurately identifying the social networks on which respondents believe they have been victims of cyberbullying is difficult due to the diversity of sites accessed by users, but also because the sources of bullying can be very diverse. On the other hand, the indication of those platforms can be confused with the platforms currently accessed, out of habit. However, the data obtained from our sample regarding the respondents’ perception of being a victim of cyberbullying and of the platforms on which this phenomenon occurs, are presented in Table 3 . Section title: Results Educational score: 0.9395471811294556 Domain: other Document type: Study Language: en Given the results presented in Table 3 , we deduce that Facebook and Instagram are the main platforms on which respondents felt that they have been victims of cyberbullying and implicitly the main platforms on which they consider that the phenomenon of cyberbullying mostly takes place. However, this results could also be influenced by the fact that these two platforms are the ones that are used most frequently globally. Hence, a previous study showed that Facebook and Instagram are indeed two of the most used social networks worldwide . In this context, considering that the interaction of people mostly takes place on these two platforms, there is a higher chance of the phenomenon of cyberbullying to also take place more often on this platforms than on other social networks. Section title: Results Educational score: 1.0190576314926147 Domain: other Document type: Other Language: en Another situation should be noted: respondents can be victims of cyberbullying (and can declare or not this) and they can also be witnesses of such situations. As we saw in the previous table, 35% of the respondents declare that they were not victims but we should rather take into account that all the subjects questioned can act rapidly for their safety. Section title: Results Educational score: 1.03335702419281 Domain: other Document type: Other Language: en Furthermore, Table 4 presents some of the forms of cyberbullying that were witnessed by the respondents. Section title: Results Educational score: 0.9784745573997498 Domain: other Document type: Study Language: en Being a witness or a victim of cyberbullying can determine various behaviors. In this regard, the results from Table 4 , showed that most respondents declared that they have seen that people who were victims of cyberbullying were mostly ridiculed with regards to their physical or intellectual aspects and features (23%). Also, other forms of cyberbullying witnessed by the respondents were the one in which the victim was verbally abused or threatened by the aggressor on social media (16%), and the one in which the victim has been excluded from a group or social circle (12%). However, 22% of the respondents declared that they have never witnessed the phenomenon of cyberbullying. Section title: Results Educational score: 0.9775186777114868 Domain: other Document type: Other Language: en In the context of taking action when witnessing cyberbullying, the list proposed in the questionnaire captures only a part of the possible decisions to be taken in such situations. In this context, by witnesses we refer to the people who witnessed cyberbullying in the case of other people. Thus, considering the diversity of the ways in which cyberbullying is defined. Thus, the courses of action taken in the case were respondents witnessed such situations are presented in Table 5 . Section title: Results Educational score: 1.01056969165802 Domain: other Document type: Other Language: en In the situations where respondents are witnesses or victims of cyberbullying, there are inevitably various ways to act starting from a set of simple questions such as: am I directly involved?; who is the aggressor?; to what extent an escalation of dialogue can be productive?; the legislative provisions related to the use of the Internet are known in detail by any user?; to what extent a whole series of activities on the Internet are considered much more dangerous? etc. All these elements can lead to the idea of a certain lack of motivation to act in cases of harassment on social media networks mainly in the quality of whiteness. In this case, is not surprising that respondents mainly chose ‘not to do anything’ in such situations (36%). Thus, only 10% of the respondents who witnessed cyberbullying got involved by making their opinion very clear to the aggressor, 8% of them sought out the victim for support/help, and 7% of them got verbally involved in the conflict, or objected to the act of harassment. Section title: Results Educational score: 1.0190587043762207 Domain: other Document type: Other Language: en However, the percentages can be spectacularly different when the respondent can be directly a victim of cyberbullying (in a hypothetical situation). In this case, the courses of action can be completely different as we it can be seen from Table 6 . Section title: Results Educational score: 1.1145371198654175 Domain: other Document type: Other Language: en Given the data presented in Table 6 , most respondents declared that if they were to be victims of cyberbullying they would report the aggressor’s account (30%), that they would confront the aggressor (27%), that they would discuss the problem with a friend (15%). However, 13% of them declared that they would ignore the situation and 10% would leave the platform. Section title: Results Educational score: 1.2726746797561646 Domain: other Document type: Study Language: en Furthermore, it is interesting to emphasise that, our research also showed that harassment situations on social networks are seen by respondents as personal rather than public or within the competence of the authorities: the students declared that in the position of victims, they want to talk with a friend (60%), a parent (19%), a relative (6%) or others, but not with teachers/tutors (just 2%). Section title: Results Educational score: 0.9713998436927795 Domain: other Document type: Study Language: en Next, we build a statistical index starting with Q11-Q18 items from the questionnaire. These items represent several eight Likert scales that summarize a series of experiences related to cyberbullying. The descriptive values of each of the eight items is presented in Table 7 . Section title: Results Educational score: 2.961071491241455 Domain: biomedical Document type: Study Language: en To all these items we applied a Principal Components Factor Analysis and the results for our sample were reliable (KMO = 0.752, p = 0.000). The results delivered a single factor (with eigenvalue = 3.29 > 1) that explained 41.23% of the variance in responses. The descriptive values of our statistical cumulative index named ‘bullying index’, are further presented in Table 8 . Section title: Results Educational score: 1.1734236478805542 Domain: other Document type: Other Language: en According to the results from Table 8 , the index has values in the interval with a mean = 12.51 and represents a useful tool to measure the extent of bullying on the Internet of our respondents. In the context of this index, we formulated the next hypothesis: Section title: Differences according to the gender of the respondents Educational score: 3.9378395080566406 Domain: biomedical Document type: Study Language: en We used the G*Power software to calculate the minimum sample volume in the case of applying a nonparametric Mann–Whitney U test for two groups. In our case, for effect size d = 0.5, alpha = 0.05 and Allocation ratio = 1, we obtained a minimum sample size of 244 subjects (sample size for each group was 122 subjects). Section title: Differences according to the gender of the respondents Educational score: 1.5062497854232788 Domain: other Document type: Study Language: en A Mann–Whitney U test was conducted and we observed that the males and females are not significantly different with the exposure to bullying on social media platforms (U = 30,275, z = −0.610, p = 0.542; mean ranks: 254.4 < 246.6). The difference between males and females is not statistically significant. The hypothesis is not confirmed. Thus, the results which showed no significant difference in exposure to cyberbullying depending on gender, might be influenced by the fact that cyberbullying is often characterized by anonymity and perpetrators may choose the victims not on the basis of their gender but on the basis of other factors, such as popularity, hobbies, or activity domain. Even more, because it takes place online, cyberbullying does not rely on force or power, like it happens in the case of face to face bullying. Hence, in the online environment the stereotypes related to man power or to the weakness of females no longer apply. Section title: Differences according to the type of studies Educational score: 3.8722927570343018 Domain: biomedical Document type: Study Language: en As in the previous case we used the G*Power software to calculate the minimum sample volume in the case of applying a nonparametric Mann–Whitney U test for two groups. In this case, for effect size d = 0.5, alpha = 0.05 and allocation ratio = 0.28, we obtained a minimum of 278 subjects (for sample size group 1 formed by undergraduate’s students) and 78 subjects (for sample size group 2 formed by master students). Section title: Differences according to the type of studies Educational score: 2.164724111557007 Domain: other Document type: Study Language: en A Mann–Whitney U test was conducted and we observed that the undergraduate students (390 respondents) and master students (110 respondents) are significantly different in the exposure to bullying on social media platforms (U = 15.950, z = −4.151, p = 0.000; mean ranks: 264.6 < 200.5). The difference between undergraduates and master students is statistically significant: undergraduate students are significantly more exposed to bullying on social media platforms. The magnitude of the effect had the value d = 0.18 so a value justifying a smaller effect size. The hypothesis is confirmed. Section title: Differences according to age categories Educational score: 3.376223564147949 Domain: biomedical Document type: Study Language: en In this case if we use G*Power software for the calculation of the minimum sample size we can use the One-way ANOVA procedure for three groups. For effect size d = 0.25, alpha = 0.05 and three being number of groups we obtained a total sample size of 252 subjects. Section title: Differences according to age categories Educational score: 3.7043545246124268 Domain: biomedical Document type: Study Language: en Because the Kolmogorov–Smirnov test of normality for the dependent variable was significant statistic ( p = 0.000) we applied a nonparametric Kruskal-Wallis Test to examine the differences in exposure to bullying on social media platforms according to age categories (1. 18–21 years, 2. 22–25 years and 3. 26–30 years old). Significant differences (Chi-square = 60.386, df = 2, p = 0.000) were found among the three categories of ages. The magnitude of the effect had the value d = 0.35 so a value justifying a medium effect size. Section title: Differences according to age categories Educational score: 2.432424306869507 Domain: other Document type: Study Language: en In the continuation of the analysis, a Mann–Whitney test was conducted to compare the group ages two by two. We observed that the respondents from the first two groups were significantly different , the first and the third group were significantly different (U = 5,375, Z = −4.884, p = 0.000; mean ranks: 180.59 > 115.69). The last two groups (2. 22–25 years and 3. 26–30 years old) were not different (U = 5,250, Z = −0.253, p = 0.800). In conclusion the first group (18–21 year old) was significantly more exposed to bullying on social media platforms compared to the second group (22–25 years old) and the third group (26–30 years old). In the same time the second and the third group are not different in the exposure to bullying on social media platforms. The hypothesis is partially confirmed. Section title: Differences according to age categories Educational score: 1.9267370700836182 Domain: biomedical Document type: Study Language: en Next, in order to test the third hypothesis of our research, we made an association analysis for the Q9 and Q19 items from the questionnaire. Section title: Differences according to age categories Educational score: 3.9302563667297363 Domain: biomedical Document type: Study Language: en In this case we used the G*Power software to calculate the minimum sample volume in the case of applying the Chi Square test of independence. This time for effect size ω = 0.3, alpha = 0.05 and df = 4, we obtained a minimum sample size of 207 subjects. This sample represents the minimum number of observations to be able to assume the statistical effect of type I error probability and power. Section title: Differences according to age categories Educational score: 1.8516792058944702 Domain: biomedical Document type: Study Language: en The results obtained from testing the hypothesis are further presented in Table 9 . Section title: Differences according to age categories Educational score: 1.1423134803771973 Domain: other Document type: Study Language: en To test this hypothesis, we analyzed the association between the dichotomous variable about the fact that the ‘harassment already began in offline’ (if no- the harassment was all the time just online) and the variable that summarise some courses of action due to harassment. If we discuss just about the respondents who were harassed before in off line (considered here 100%) we obtained that just 30% declared that they ignore the situation, 50% want to leave the platform, 48% declared that they want to confront the aggressor, 26.7% intend to report the aggressor’s account and just 26.7% talk with friends about the course of action. All the values until 100% are reserved to the course of action if the harassment is online. Section title: Differences according to age categories Educational score: 1.4287630319595337 Domain: other Document type: Study Language: en We observed from Table 9 , that the association of the two variables was statistically significant ( χ 2 (4) = 22.228, p = 0.000). Thus, we can conclude that the courses of action chosen by a possible victim of on-line harassment are significantly statistically different. For example, the respondents would rather report an account if the harassment did not start in the offline environment. Thus, the respondents would report the account if the harassment started online. Also, most respondents will ignore the situation if the harassment starts directly online. In other words, most respondents will ignore the situation if the harassment does not start in the offline environment. The hypothesis was confirmed (the association was on the low level of intensity phi = 0.216, p = 0.000). Section title: Differences according to age categories Educational score: 0.997874915599823 Domain: other Document type: Other Language: en Furthermore, in the context of preventing or combating the phenomenon of cyberbullying, when asked about the actions that should be taken in this regard, respondents proposed some solutions which refer to: blocking the account of people who use insults, threats or inappropriate words towards other people, reporting the account or the aggressor to the authorities, informing and teaching people about the existence of this phenomenon so that they could know better how to deal with it if they experience or witness it. Section title: Discussion and conclusions Educational score: 0.9711006879806519 Domain: other Document type: Study Language: en The development of social media platforms facilitated the communication and interaction between people, but also led to the development of cyberbullying—bullying in the online environment. In this context, the purpose of our paper was to analyze how the phenomenon of cyberbullying manifests in terms of frequency on social media platforms, while taking into account factors such as gender, and elements related to the behaviour/reactions of witnesses and victims. In this regard, the objectives of the research referred to: establishing the main form of cyberbullying that the investigated population has most often faced on social media, determining which of the genres was more exposed to the phenomenon of cyberbullying, establishing to what extent the students were aware of the presence of the phenomenon on social media networks, and determining how often the respondents use social media platforms. Section title: Discussion and conclusions Educational score: 1.0521466732025146 Domain: other Document type: Study Language: en Considering the objectives of the research, the results showed that most respondents use social networks often and very often, and that the main forms of cyberbullying experienced or witnessed by students on social media platforms are represented by: ridiculing people’s physical or intellectual aspects and features, being verbally abused or threatened, being humiliated by posting disturbed/sensitive images and content, being excluded from a group, or having their secrets publicly revealed. From this perspective, our paper is in line with other studies which described the forms in which cyberbullying is manifested . Moreover, our paper is in line with a previous study conducted on Romanian students , which showed that respondents declared that they have encountered cyberbullying in the form of spreading rumours, in the form of being banned or excluded from groups or by being humiliated online. Section title: Discussion and conclusions Educational score: 1.0523724555969238 Domain: other Document type: Study Language: en Furthermore, the results showed that most respondents were aware of the existence of the phenomenon of cyberbullying on social media, and the results showed there were no differences found in exposure to bullying on social media according to gender. However, a previous study conducted at the level of students from Romania , showed that the respondents believed that females were more prone to being victims of cyberbullying, compared to males. Contrary to the results of our paper, a previous study conducted on Canadian students, showed that cyberbullying was influenced by the gender of the respondents and that same gender bullying was more often experienced than opposite gender bullying. In other words, females felt that they were more bullied by other females and males that they were bullied by other males. Even more, the same study also showed that females reported more negative effects of cyberbullying on their personal life and on their educational performance, compared to male respondents . Section title: Discussion and conclusions Educational score: 1.2817728519439697 Domain: other Document type: Study Language: en Given the first hypothesis of our research, the results revealed that younger students (those aged between 18 and 21 years old) tend to be more exposed to online bullying, compared to students aged between 26 and 30 years old. Thus, the first hypothesis was confirmed. Section title: Discussion and conclusions Educational score: 1.2402042150497437 Domain: other Document type: Study Language: en Moreover, in the case of the social networks on which the respondents declared that they have been victims of cyberbullying, or on which they considered the phenomenon to be most present, the main social networks mentioned were Facebook and Instagram. In this regard, our study is in line with a previous research conducted on students in the United Arab Emirates , in which Facebook and Instagram were identified as the main platforms on which cyberbullying takes place. However, this result could also be influenced by the fact that Facebook and Instagram are two of the most used platforms worldwide, and this would mean that the increased frequency of cyberbullying on these social networks could be given by people’s frequency of using them. From this point of view, our paper is in line with previous studies , which stated that together with the increase of the numbers of users of social media platforms, an increase of cyberbullying can also occur, due to the fact that aggressors can identify their potential victims more easily. Even more, our paper is also in line with a previous study which was conducted on young people aged 14 to 17 years old from seven European countries , which emphasized the fact that in the context of Romania, Poland and Germany, spending more than 2 h per day on social networks was associated with a higher risk of experiencing cyberbullying. Section title: Discussion and conclusions Educational score: 1.0181894302368164 Domain: other Document type: Other Language: en Given the actions that the respondents took in the case in which they witnessed cyberbullying, a rather concerning results showed that most of them declared they did not take any action. In the event in which they took action, some of them actions taken referred to making their opinion clear to the aggressor, getting verbally involved in the conflict, or objecting to the act of harassment. Section title: Discussion and conclusions Educational score: 1.0512914657592773 Domain: other Document type: Other Language: en However, if they were to be victims of cyberbullying, students declared that they would mostly report the aggressor’s account, they would confront the aggressor, or they would discuss the problem with a friend. Even more, most students would discuss the issue with people they hold close, but they would not discuss it with their teachers. Section title: Discussion and conclusions Educational score: 1.1477893590927124 Domain: other Document type: Study Language: en Considering the second hypothesis of our research, the results showed no differences in the degree of exposure to cyberbullying between males and females, but they revealed differences according to their level of study and the age of the respondents. In this regard, undergraduate students were more exposed to online bullying compared to master students, and students aged between 18 and 21 years old were more exposed compared to those aged between 22 and 25 years old or between 26 and 30 years old. Hence, the second hypothesis was partially confirmed. Section title: Discussion and conclusions Educational score: 1.0980679988861084 Domain: other Document type: Other Language: en Considering the third hypothesis, the results revealed that the actions taken by students in the event of bullying are different depending on the environment in which the harassment has taken place (online or offline). Thus, students would report the account if the harassment started online, but they would ignore the situation if the harassment started directly online. Section title: Discussion and conclusions Educational score: 0.978241503238678 Domain: other Document type: Study Language: en Furthermore, in the context of preventing the development of cyberbullying, some of the solutions proposed by the respondents included: blocking the social media accounts of people who address insults or abuse other people online, reporting their accounts to the authorities or educating people about the existence of the phenomenon of cyberbullying and its consequences. In context of educational measures, our paper is in line with a previous study , which emphasized the need for internet safety education and for the education of witnesses or third party observers of online bullying. In the context of solutions which could be taken into account for the prevention of cyberbullying in educational institutions, some digital education programs could be developed, and some new features could be added to the social media platforms, feature which could help with the detection of aggressive behaviour. Even more, institutions could develop mentorship programs for students, in which older students could help younger students to use social media in a more responsible manner and to protect themselves from cyberbullying and its negative effects. Taking into account the theoretical and practical implications of our paper, from a theoretical point of view, the paper contributes to the literature on the forms of manifestation of cyberbullying on social media platforms, from the perspective of students. Given the practical implication of our paper, the results highlight the need of including information about the phenomenon of cyberbullying in the education of young people, the need of conducting awareness and prevention campaigns or programs. Even more, the paper highlights the need of developing and implementing instruments on social media platforms, that could detect the improper behavior of users. Thus, in the context of educators, the results of the study could be taken into account by educators and teachers, and they could be presented to high school students and university students in order to increase awareness about the phenomenon of cyberbullying. Furthermore, policymakers could make use of the findings of our study by looking at the forms of cyberbullying encountered by students, and at the platforms on which this phenomenon is most likely to take place, and they could develop policies which could contribute to diminishing this phenomenon. Next, social platforms could take into account the findings of the research in order to develop policies against cyberbullying on social networks, or in order to create and develop filters/buttons which could help users report bullying or stop user from bullying each other. Section title: Limitations and future research directions Educational score: 1.0571306943893433 Domain: other Document type: Other Language: en Considering the limitations of our research, one limitation is represented by the fact that only quantitative methods were used in order to conduct the research. In this regard, a future research could take into account using qualitative methods, such as interviews in order to gather information about the phenomenon of cyberbullying. Another limitation is represented by the fact that data was obtained from students only from one university, and in a future research, the opinion of students from other universities from Romania or abroad, could be studied. Moreover, apart from gathering information from students, in a future research, the opinion of decision makers or representatives of authorities could be taken into account, in order to analyze ways in which cyberbullying could be prevented or combated. Other research directions could be represented by analyzing the role of artificial intelligence in preventing cyberbullying, and on comparing cyberbullying with forms of traditional bullying in order to better develop strategies or policies meant to prevent the development of such phenomenons. | Other | other | en | 0.999997 |
PMC11695935 | Section title: 1 Introduction Educational score: 1.0246766805648804 Domain: other Document type: Other Language: en In the British journal “The Economist” there is a column on management which is named “Bartleby”. On 18 September 2023, Bartleby undertook an interesting thought exercise by asking: “ Who is the most important person in your company? ” Section title: 1 Introduction Educational score: 1.3030532598495483 Domain: other Document type: Other Language: en The search for an answer to the above question in a research-based pharmaceutical company might lead to a scientist who invented a new product, which became instrumental in securing the presence and future of the company. However, this would not be the usual answer! Section title: 1 Introduction Educational score: 1.225212812423706 Domain: other Document type: Other Language: en In research-based pharmaceutical companies, the CEOs and those who have the ear of the boss on important issues (the so called “Panama Canal people”) are the most important people, having great decision-making power and long-term impact. They, therefore, have the power to take decisions about initiation, continuation or discontinuation of projects, thereby on short-, medium- and long-term research and development in the company. However, as a rule, these are marketing, business and financial experts. Beyond industry, no one would think that, for example, the commercial director of a university hospital or of a large research institute would have the final decision-making power over the various research and development projects. In the industry, however, this is normal. The CEO and the Executive Board determine the direction of a company, which in effect leads to management announcements such as: Section title: 1 Introduction Educational score: 1.1576377153396606 Domain: other Document type: Other Language: en “Our company plans to advance a more productive pipeline for new therapeutics using lessons gained from recent research setbacks with the guidance of a new management team. The goal is to accelerate decisions on high-risk, high-reward projects, because we need to deliver value fast, and that’s the mentality we”. Section title: 1 Introduction Educational score: 2.5357532501220703 Domain: biomedical Document type: Other Language: en It is easy to conclude that both the participation of scientists in executive boards and a suitable training and qualification of CEOs and members of the executive board would be required to make research-based pharmaceutical companies more efficient and successful for achieving new innovative medicines and to optimize the huge financial investments in industrial drug R&D. In this paper we try to point out why this is crucial: • What are the core competencies and soft skills of CEOs and executive board members in research-based pharmaceutical companies? • How should top managers of research-based pharmaceutical companies be trained and educated top-down or bottom-up? Benefits for R&D staff? • What might a certification course include in order to entitle managers to lead such a company? PharmaTrain Syllabus (v 3.0) already provides the required tools ready for adaptation to CEO training. Section title: 1 Introduction Educational score: 1.351944088935852 Domain: other Document type: Other Language: en Our proposals on how to achieve optimization of R&D processes in pharmaceutical companies does not focus on the staff working in laboratories, clinics, project groups, controlling departments, etc., as it is usually done, when R&D processes are to be optimized. We focus on the role of CEOs and executive board members, currently and in future. We feel that this is an aspect that has not yet gained sufficient attention until now. CEOs and executive managers usually initiate the optimization of business processes in their organizations, and thus have an impact on research and development. They steer and define such processes but are not themselves subjected to the process . CEOs are usually not blamed for mistakes, and they often have no notion that a failure in the R&D departments could have been based on their decisions. Section title: 2.1 Goal - Successful drugs Educational score: 1.4038978815078735 Domain: biomedical Document type: Other Language: en What is the goal of a pharmaceutical company? It is to be successful in marketing the most effective medicinal products. However, what is a successful drug? Which key criteria must be met? Section title: 2.1 Goal - Successful drugs Educational score: 3.769623041152954 Domain: biomedical Document type: Other Language: en Key criteria for a successful drug: 1. High medical need for an important therapeutic goal. Not today but in about 5 to 10 years! 2. Intellectual Property: patent protection for as long as possible and marketing authorization as early as possible. Investments made for such projects, but also for those projects that were carried out in parallel but failed, must flow back as income and must generate an overall profit for the company. 3. Innovative approach: accepted technology and scientifically accepted mechanism of action, which includes understanding the targeted disease. 4. Efficacy: Improved therapeutic efficacy for targeted medical indication. 5. Safety: Better benefit-risk ratio than existing pharmaceuticals. 6. Production: Favorable cost-benefit ratio, cost accepted by state health systems. 7. Regulatory approval: the new drug must meet the standards set by international guidelines and by competent authorities, such as the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA) in Europe. 8. Drug outcomes: “Efficiency” for the targeted indication under real-life conditions . Section title: 2.1 Goal - Successful drugs Educational score: 1.6050275564193726 Domain: biomedical Document type: Other Language: en These points are well known, and numerous excellent experts have established optimized approaches in their R&D organizations. The overall success of the pharmaceutical industry, however, is modest. The vast majority of R&D projects in the pharmaceutical industry, despite huge financial and resource investments as well as continuous process optimization, cannot be successfully completed. Only a very small fraction of initiated projects turns out to be a real economic and medical success. Section title: 2.1 Goal - Successful drugs Educational score: 1.8272781372070312 Domain: biomedical Document type: Other Language: en CEOs and board members are meant to be competent for the first two key criteria. There is no special thinking, language or training in place for e.g., EU and EFPIA to jointly them to handle most important issues, e.g., improvement of research, removing development bottlenecks in the drug development process, or (dis-) continuation of an R&D program–in a given case. R&D people, regulatory experts and specialists in synthesis, production, quality, safety, efficacy and (post-)marketing are expected to cover key criteria numbers 3 – 7, but also 1, 2 and 8. Specific trainings are available to them, and can be found as summarized in the PharmaTrain Syllabus 3.0 (see Supplementary Material ). Such trainings include learning about thoughts, culture and language/thesaurus of colleagues, who have developed these processes separately. Section title: 2.1 Goal - Successful drugs Educational score: 1.8179547786712646 Domain: biomedical Document type: Other Language: en The PharmaTrain Syllabus is one result of the first Innovative Medicines Initiative (created by the late Prof. Fritz Bühler), a joint project of the European Commission and EFPIA–guaranteeing participation of the best of the best for deriving best possible answers–case by case and in general. It was set out to fully grasp all developmental details of pharmaceutical R&D, laying out principles in a language common to all concerned. It encompasses 13 Sections starting with drug discovery and ending with health economics, modern outcomes research and issues on patient access (for further details see Supplementary Material ). Section title: 2.2 Drug research and development (R&D) politics Educational score: 2.2565855979919434 Domain: biomedical Document type: Other Language: en It is often said that innovation cycles in medicine are constantly accelerating, however, this is only true for the applied technologies. The medical innovation, for example, the approval of new innovative medicinal drugs, lags behind. One example is Alzheimer’s disease, for which, despite enormous efforts, no scientific breakthrough, that would succeed in stopping or significantly slowing down the progress of the disease had been achieved for a long time. Contradictory decisions by FDA and EMA concerning lecanemab show that high expectations on efficacy and efficiency are in reality small and risky. Such are difficult to understand and accept by CEOs, R&D management, and patients concerned. Section title: 2.2 Drug research and development (R&D) politics Educational score: 1.9968146085739136 Domain: biomedical Document type: Other Language: en New software solutions, including simulation and automation technologies, appear frequently on the market and are always welcomed with much advance praise and high hopes. After some time, they are replaced by new procedures, usually, when it becomes clear that biological complexity prevented the intended solution, and the costs had increased enormously. As a matter of fact, the perceived complexity of biology does not decrease with new scientific discoveries but is often increased. Section title: 2.2 Drug research and development (R&D) politics Educational score: 3.402378797531128 Domain: biomedical Document type: Other Language: en To optimize the R&D process, the management of most companies and the additionally committed consulting firms work intensively to be able to efficiently control the R&D process. To be able to control the R&D process efficiently, tools are required, which should allow a reliable measure of progress and its value. According to current management rules and those of frequently involved consultants, managers should be able to define which measures lead to success and which do not. For this purpose, so-called key performance indicators (KPIs) have been defined with which the success of measures can already be determined after a short time. The big question is which measurement parameters are meaningful regarding drug R&D. One of the principal problems measuring KPIs in pharma R&D is the very long period between the initial investments and the finished product, but there are also other principal problems, like the specific character of drug R&D projects in an area of tremendous biological complexity. From popular publications it was stressed that there is one exemption from this rule: mRNA-based vaccines for COVID-19. But as we know from the Nobel Prize in medicine in 2023 that Katalin Kariko and Drew Weismann had started their research 30 years ago: the toolbox needed was ready and available for use in developing anti-COVID vaccines almost on time. Such development will not happen soon again, even for those drug candidates using the same technology platform. Section title: 2.2 Drug research and development (R&D) politics Educational score: 1.4024945497512817 Domain: other Document type: Other Language: en The very long period of development and the frequent data-driven changes in the R&D process make it difficult to measure the performance of an R&D organization in the way people are used to from other business and production areas. Drug R&D is one of the most technology-intensive industries, investing many billions of euros every year. It not only fails to deliver more success from investment but also lags behind the innovation performance of other industries. The current trend for new drug approvals shows an even further decrease. Section title: 2.2 Drug research and development (R&D) politics Educational score: 2.8142950534820557 Domain: biomedical Document type: Other Language: en Training of regulators and researchers is aimed especially at dealing with the frequent changes resulting from study outcomes, e.g., non-clinical and clinical results. The impact of missing or dangerous information in all subsequent steps as well as previous ones can be found in the PharmaTrain syllabus. Whilst CEOs and managers do not need to understand each and every detail, they should be capable of understanding and discussing single aspects as they arise case by case. Such aspects can be taken from the tables published by K. Olejniczak . Here, CEOs, regulators and scientists can sit together to understand the need or not e.g., of reproductive toxicity testing at a certain stage of drug development derived from risk assessment and mitigation, and the possible impact on overall development–when such toxicity would be expected to possibly happen. It is the crux of medicines development that upper management decisions are often taken without understanding the real risk issues and their probabilities . There is no quick fix, but regulators and scientists should be alert for knowledge deficiencies of CEOs and upper management and urgently recommend special trainings. Again, the catalogue of PharmaTrain contains a complete compilation of potentially upcoming issues. Section title: 2.3 Drug research and development (R&D) management strategy Educational score: 1.5188645124435425 Domain: other Document type: Other Language: en Managers and consulting companies try to take lessons learnt from more successful and faster industries to improve and accelerate the drug R&D process. According to common management theories, R&D functions must work closely with the commercial and corporate strategy teams to understand what the company wants to achieve. The R&D team should be guided by the priorities of the company and at the same time make clear what is scientifically and technically possible and feasible, today also including the use of artificial intelligence (AI) in clinical trials, pharmacovigilance, etc. The R&D strategy and corporate strategy must be aligned to answer questions such as: What are the company’s goals? Which R&D activities are required to achieve them? Section title: 2.3 Drug research and development (R&D) management strategy Educational score: 1.3227345943450928 Domain: other Document type: Other Language: en Aligning the two above mentioned strategies is not easy. Usually in companies, the corporate strategy is set out prospectively in a five-year plan or covers even shorter periods. However, this is too short a time horizon to manage R&D in the pharma industry, where the product development period is much longer. To make this first step right, corporate strategy leaders should actively engage in research and development, which also requires a deep understanding of the relevant scientific background. Section title: 2.3 Drug research and development (R&D) management strategy Educational score: 1.3783667087554932 Domain: other Document type: Other Language: en This happens at a time, when no details on go/no go decisions during later development are available. Therefore, corporate strategy leaders should have a basic understanding of R&D opportunities and potential pitfalls. Whereas the PharmaTrain syllabus encompasses all possible aspects, it cannot be expected that a full training will be taken by all CEOs and controlling managements. From our point of view such training is highly recommended, and will benefit in “developing better medicines faster” (slogan of PharmaTrain). Section title: 2.4 Drug research and development (R&D) technical prerequisites Educational score: 3.3321733474731445 Domain: biomedical Document type: Other Language: en An important prerequisite is digital empowerment, which today affects almost every aspect of the R&D function. Artificial intelligence can be used primarily in the early stages of projects to help identify new biological mechanisms or new applications of existing technologies. Based on this, automated screening procedures with high throughput can help to carry out a selection of possible candidates. Digital simulations can be used if no substances have yet been synthesized. Digital communication tools address the connectivity issues that occur with geographically distributed project teams, as is normal in large pharmaceutical companies. These tools have become indispensable for bringing together the often geographically distant employees in order to exploit synergies. However, truly innovative ideas often arise in direct personal encounters between people. The serendipity of chance encounters can be one of the conditions of many breakthrough innovations. Whether this can be generated significantly with so-called innovation centers has not been shown until now. Section title: 2.4 Drug research and development (R&D) technical prerequisites Educational score: 1.7618027925491333 Domain: other Document type: Other Language: en Most importantly, developing a strategy for pharmaceutical R&D involves some unique challenges that other industries do not face. On the one hand, scientists and regulatory employees have to take decisions that go far beyond their scientific or regulatory core competences, such as customer, market and economic factors–and these decisions may have consequences much longer than the 5-year business plan of the company. On the other hand, stakeholders, up to the senior managers, governed by company business criteria and residing outside the R&D laboratories, have to understand complex technologies and development processes and even more complex biological relationships and think about a much longer time horizon than they are accustomed to, before overriding the decisions of scientists and regulators. Section title: 2.4 Drug research and development (R&D) technical prerequisites Educational score: 1.2398148775100708 Domain: other Document type: Other Language: en General and specific needs and opportunities for training CEOs and senior managers can be taken from decision-tree risk strategies , and from the PharmaTrain Syllabus v 3.0, 2024 . It should be stressed that the need for training arises at specific and unforeseen decision points, calling for action and training by demand and on the spot. To avoid errors from fast trouble shooting, long-term full training is preferred. Section title: 2.5.1 Steering R&D processes Educational score: 1.3836357593536377 Domain: other Document type: Other Language: en Among many other questions, the failure of the majority of R&D projects raises one big question: Why is steering drug R&D processes and projects so difficult? Section title: 2.5.1 Steering R&D processes Educational score: 1.2607159614562988 Domain: other Document type: Other Language: en Of course, this question is not new, and answers have already been taught by business schools or international consulting companies. Pharmaceutical companies invest considerably to learn from these organizations on how to improve and to increase the outcomes quantitatively and qualitatively. One basic problem answering this question and finding an effective solution is the available database. No company, neither in the case of success nor failure, honestly and transparently states what has finally led to success or failure. On the contrary, the reason behind a success is usually kept confidential inside the company and the true reason for a failure is kept obscured so that the company does not suffer any damage to its image. At the least, no systemic errors should become public. All companies are obliged to the latter, out of consideration of the shareholder value. Section title: 2.5.1 Steering R&D processes Educational score: 1.298479676246643 Domain: other Document type: Other Language: en Nevertheless, there are a number of questions that need to be answered. A selection of such questions could be: 1. Which project organization is optimal for R&D projects in pharma? 2. Which procedures, processes and communications are needed? 3. Which mindsets are crucial at the different hierarchical levels? 4. Which qualifications of the people involved are needed or optimal? 5. Which type of controlling is needed for not obstructing sensitive innovation processes? 6. Which type of funding is needed for a learning process? R&D projects are learning processes, and any day can deliver unexpected data which may force a complete change of the plan! Section title: 2.5.2 Failure of R&D processes Educational score: 1.1094214916229248 Domain: other Document type: Other Language: en The fact that high-tech R&D projects may fail is well known in industry and in research institutions. The reasons are often similar: mostly insufficient communication with potential customers, over-optimistic targets and ballooning costs, resulting in the product not being sold on the market. Section title: 2.5.2 Failure of R&D processes Educational score: 1.4494930505752563 Domain: other Document type: Other Language: en The advice given could be for example,: use an intelligent project management software and with this support, the projects will stay on track and will deliver on time and under budget. Successful projects begin with a good plan, but it may be hard to keep all necessary tasks under control. Many tasks may be dependent on others and therefore across the timeline. An IT tool may provide important help, e.g., by breaking the project into intelligent milestones. Such tools may be a decisive help for high-tech projects, and they may also be helpful for project managers in drug R&D, however, the problem with drug R&D is that a project involving biology is extremely complex. Section title: 2.5.2 Failure of R&D processes Educational score: 1.3611164093017578 Domain: other Document type: Other Language: en A drug R&D project is a continuous learning project. The project team can go forward only step by step, adhering more or less to the straight initial outline of R&D. Every day results can be obtained which are, firstly, unexpected and, secondly, can cause a completely new situation. One question is always: is the organization’s strategy balanced between committed decisions and flexibility and learning? R&D strategies have very long-time horizons when it comes to real innovation and not just incremental innovation. Section title: 2.5.2 Failure of R&D processes Educational score: 3.5656912326812744 Domain: biomedical Document type: Other Language: en The development of a cancer drug can take more than 15 years and in the end the product has failed in oncology but may be approved for a completely different serious disease or may have proven to be a complete flop. In drug research and development, it is almost the rule that a therapeutic indication (project goal) has to be changed during the R&D process or proves to be completely impossible, e.g., because of side effects in the complex biological system that had not been predictable. A drug against depression or schizophrenia needs to consider that the human brain manages 10 trillion (10 13 ) calculations in one second! The question to the researchers: Which one of those steps is going wrong and what happens to the neuronal network if one step is changed by a certain drug compound? Section title: 2.5.2 Failure of R&D processes Educational score: 2.7411391735076904 Domain: biomedical Document type: Other Language: en Prospectively, it is very important to understand that only very seldomly the therapeutic target indication on which a drug R&D project was started, is exactly that approved for the market. It, therefore, remains a central task of R&D departments, in the event of the failure of the original target indication, to try to extract a new therapeutic option for the project from the data accrued. This is important not only for financial, but also for scientific and ethical reasons because animals and humans treated up to this point with the drug candidate should not have been used in vain. The data already compiled is usually very valuable. Companies have been established which have specialized in developing successful drugs from failed projects of other companies (drug repurposing). Section title: 2.5.2 Failure of R&D processes Educational score: 1.2903176546096802 Domain: other Document type: Other Language: en A number of top drug repurposing companies can be found here: https://www.ventureradar.com/keyword/drug%20repurposing . Section title: 2.5.2 Failure of R&D processes Educational score: 1.665053129196167 Domain: other Document type: Other Language: en In this context, it should also be pointed out that a fundamental difficulty of drug R&D is to predict which type of drug will have a therapeutic and economic success 15 years from now. It is not known today what will really be needed in 15 years, and secondly, which type of drug will make a splash in the pharmaceutical market. Marketing departments do not know this either, because they analyze the current market and determine which blockbuster product the company is missing on the day after tomorrow. Section title: 2.5.2 Failure of R&D processes Educational score: 1.8692747354507446 Domain: biomedical Document type: Other Language: en Based on current new findings, research departments are trying to anticipate the future and dream of developing successful or even blockbuster drugs. However, one must not forget that truly innovative projects require a change in thinking and therefore always experience effective internal opposition. In addition, doubters are usually viewed as very intelligent, while enthusiastic followers of an idea are quickly viewed as naive and gullible. Drug research remains a bet on the future, because despite the help of modern techniques, human beings have a fundamental problem: they cannot reliably predict the future and are not able to include all eventualities in planning a project where on any day anything may happen. Even the annual budget planning for R&D projects remains an attempt to pretend to have the next year of the drug R&D projects under control. When doubt proliferates it becomes dangerous for an innovative project, especially when it proliferates into the executive management. Section title: 2.5.2 Failure of R&D processes Educational score: 1.1712099313735962 Domain: other Document type: Other Language: en Despite all this, executive leaders must devote significant funds to drug research and development (R&D) even though such investments are risky, their potential less visible to the stakeholders and the public compared to many other investments, and typically bear fruit only after the executive manager has already left the company. Executive managers are under great pressure. Section title: 2.5.2 Failure of R&D processes Educational score: 1.7834293842315674 Domain: biomedical Document type: Other Language: en Although most drug developments are terminated during clinical development or toxicology, this may also happen for non-scientific reasons. According to the rules of responsible management, it is important to stop an expected to fail project as quickly as possible, even if the developing researchers might vote to continue development. Section title: 2.5.3 Leadership qualification for R&D based pharmaceutical companies Educational score: 1.4004520177841187 Domain: other Document type: Other Language: en In order to achieve a more successful industrial development of innovative drugs, the top managers of the companies should specifically be trained and educated for their specific task. So far, only the scientists and managers directly involved in research are fully trained bottom-up to generate more successful R&D drug projects. This is still important, but not only purposeful, because an additional tailor-made training and education system should also be set up top-down for executive managers! Section title: 2.5.3 Leadership qualification for R&D based pharmaceutical companies Educational score: 1.4425662755966187 Domain: other Document type: Other Language: en In the healthcare sector, especially in the field of prescription-only drugs, in contrast to the normal market economy, it is not the individual consumer (patient/organization) who decides on the purchase of a certain product and on the question of whether the product is allowed to enter the market at all and to remain there. In addition, enormous public funding is being used in healthcare. The higher these contributions rise, the more it must be expected that calls for a communitarisation of certain parts of the system, for example, the pharmaceutical industry, will become louder and louder. The pharmaceutical industry is traditionally seen as an industry that makes high profits at the expense of society. Section title: 2.5.3 Leadership qualification for R&D based pharmaceutical companies Educational score: 1.4484490156173706 Domain: other Document type: Other Language: en Balancing R&D costs versus the economic benefit (also under the pressure of the stakeholders) needed to further development of next generations of medicines needs to be communicated well. Communitarisation as practiced previously in the Eastern bloc did not yield better results. Section title: 3.1.1 Core competencies and soft skills Educational score: 1.266937255859375 Domain: other Document type: Other Language: en Executives need excellent management skills and leadership capabilities, however, contrary to pharmaceutical companies in the generics field, executives in research-based pharmaceutical companies should have the mindset of real entrepreneurs. They should not just be business and marketing experts. Which means that they should primarily be proud of their competent employees (including researchers) and their excellent drug products and only secondarily of profit. If a company creates excellent drug products, profit comes automatically! Section title: 3.1.1 Core competencies and soft skills Educational score: 1.599904179573059 Domain: other Document type: Other Language: en Regarding Pharma R&D projects, modern management tends to expect well-structured or even linear narratives as in high-tech projects. In other words, there is a belief that after a series of intense scientific challenges, the obstacles of a project may culminate, but in the end good management leads to a successful result. Therefore, in case of failure of R&D projects, the responsible management and the research groups involved are considered to be substandard. Section title: 3.1.1 Core competencies and soft skills Educational score: 1.677949070930481 Domain: biomedical Document type: Other Language: en Reality, however, is somewhat different and by no means corresponds to these presumed linear narratives. This means that a senior manager in pharmaceutical R&D can do everything right but will still be at the mercy of biological complexity, which is orders of magnitude higher than people can imagine. In other words, nature has to play along for success. The manager has to be guided by science, this knowledge should lead to increased imagination and openness for alternatives, but then more importantly, the manager should have the strength to stick to it, even if the situation does not look promising in the short term. Section title: 3.1.1 Core competencies and soft skills Educational score: 1.1558507680892944 Domain: other Document type: Other Language: en Executives should have their own vision of what the company wants to achieve to contribute to medicine and medical needs, and how they would define leadership, to lead their employees to commit to this goal. Section title: 3.1.1 Core competencies and soft skills Educational score: 1.0868068933486938 Domain: other Document type: Other Language: en Executives should be keen to learn what is seen differently by other people in the company, including the capability of self-criticism. Self-criticism and an open mind are basic requirements for scientists, therefore for an executive manager of a science-based company this should also be one important skill, which could be improved by further training options. Section title: 3.1.1 Core competencies and soft skills Educational score: 1.5533788204193115 Domain: other Document type: Other Language: en Executives should be keen to learn from national and international competent authorities as to their perception of what type of drug should be developed and which support these authorities could give during the research and development process. In other words, a real dialogue should occur on a high level and not just the formal scientific advice which is defined in the guidelines. Of course, the formal scientific advice remains key for the project groups to support a proper development of high-quality medicines which are effective and safe. Section title: 3.1.1 Core competencies and soft skills Educational score: 1.0055638551712036 Domain: other Document type: Other Language: en Executives should have the courage to be trendsetters instead of being managers that would always do what executives of competitor companies are doing. To do what executives of other companies are doing means to minimize their own personal risk. In other words, if a CEO makes the same mistake as any other CEO, he doesn’t make a mistake. On the other hand, if a CEO tells the shareholders and investors that he or she wants to do everything differently than the others, he or she takes a very high personal risk. Motto: whoever does what everyone does, risks nothing, even if it is possibly wrong. If the subordinate hierarchy levels duck and also agree, the impression of making the right decision is further reinforced and this would be the safest way to take a wrong decision. Section title: 3.1.1 Core competencies and soft skills Educational score: 1.0797127485275269 Domain: other Document type: Other Language: en Executives should not concentrate on cost control (controlling). They must create mutual trust regarding the use of company resources and should leave considerable freedom to their innovative coworkers in research and development. Section title: 3.1.1 Core competencies and soft skills Educational score: 1.0550322532653809 Domain: other Document type: Other Language: en Executives must give the scientist the feeling that they are really interested and engaged in their projects. This can be achieved by regular and in addition spontaneous communication. A show of emotion is allowed, e.g., empathy, but also respect for other opinions combined with self- and social awareness is necessary. Section title: 3.1.1 Core competencies and soft skills Educational score: 1.2035980224609375 Domain: other Document type: Other Language: en Executives of research-based pharmaceutical companies need to be prepared for undesirable news coming from the projects. They must know how to properly respond to such news. Section title: 3.1.1 Core competencies and soft skills Educational score: 1.0731666088104248 Domain: other Document type: Other Language: en Executives must also be able to inspire, otherwise they will not be able to raise funds. Section title: 3.1.1 Core competencies and soft skills Educational score: 1.1081749200820923 Domain: other Document type: Other Language: en When developing the R&D strategy and steering R&D projects, it is important to consider which competencies already exist in the company and which still need to be developed. For example, are there competent executive employees (including board members and CEO) with overarching skills and working methods that can develop and manage such an organization? Which new and important technologies could be acquired by buying another company? Section title: 3.1.2 Top-down training and education of executive managers Educational score: 1.03445565700531 Domain: other Document type: Other Language: en As pointed out, short-term decisions of executive managers have far-reaching and long-lasting consequences not only for economic and marketing issues, but also for the companies’ R&D departments and projects! However, present mutual misunderstanding between R&D and top management is common as both sides are using a different language and thinking. The two sides are talking past each other, though both are making valid points. Section title: 3.1.2 Top-down training and education of executive managers Educational score: 1.2025505304336548 Domain: other Document type: Other Language: en Researchers, to be scientifically successful, cannot change their scientific language and thinking (“subject-matter expertise”). Therefore, top managers should learn and understand R&D people. Executive managers must understand the language and the thinking of R&D people because this is the only way to build up knowledge regarding the real status of the company’s R&D projects and to take informed decisions. Section title: 3.1.2 Top-down training and education of executive managers Educational score: 2.509416341781616 Domain: biomedical Document type: Other Language: en A new thinking is going to take place: in former times in pharma industry the focus was always on developing “blockbusters”; thousands of patients should be treated repeatedly for long times. The antineoplastic agent = Kymriah, developed by Novartis, is a personalized T- cell therapy. The antineoplastic function can be successful through a single infusion, and the treatment success holds a probability today of 80%. This treatment costs 475,000 dollars for one patient. Novartis decided that patients should only pay for this drug when the treatment is successful. This “outcome–based” principle is absolutely new. The concept is altogether revolutionary compared to our traditional thinking for strategies in drug development. Section title: 3.1.2 Top-down training and education of executive managers Educational score: 1.0652145147323608 Domain: other Document type: Other Language: en Executive board members should therefore be forced to review their thinking and complete a kind of executive education program tailored to their specific needs. Trainers could be still active or former executive experts from different companies and authorities that possess much experience regarding science and regulation but would no longer be pursuing a professional career (e.g., pensioner), as they must be independent from management. Section title: 3.1.2 Top-down training and education of executive managers Educational score: 1.736615777015686 Domain: biomedical Document type: Other Language: en To build up such knowledge, a suitable basis could be taken from the content as taught and tested in PharmaTrain, which is used all over Europe, e.g., in the master courses of MDRA (Bonn University) and EUDIPHARM (Lyon University). ECPM (University of Basel) has already trained - in the master course in Medicines Development CEOs of small and medium sized companies . For a list see the Supplementary Material . The details to be offered for training CEOs and board members are given in the Supplementary Material . For discussion and decision making, the flow charts developed by Olejniczak present a very good opportunity to include all concerned in deriving at proper and defendable decisions. Section title: 3.1.3 Certification Educational score: 1.3283251523971558 Domain: other Document type: Other Language: en A certification which would specifically entitle and qualify managers to lead a research-based pharmaceutical company can be easily derived from PharmaTrain and the Syllabus developed there (for details see Supplementary Material ). The Syllabus is applied by a number of universities for certification and university degrees (e.g., Basel, Budapest, Lyon), and is being used by the University of Bonn for their course - and the authors are part of the teaching staffs. Section title: 4 Discussion and conclusion Educational score: 1.1684459447860718 Domain: other Document type: Other Language: en Many of so-called failed projects in drug R&D seem to fail because the decision to change the path of R&D is taken by the executive management. Such should be a good reason to reflect on possibilities to put this part of the process under a kind of quality check. In the usual quality assurance process, each and every person involved is checked on their level of qualification. The question always asked is: “Is the qualification level sufficiently high with regard to the task to be performed?” Section title: 4 Discussion and conclusion Educational score: 1.3724422454833984 Domain: other Document type: Other Language: en As executive managers, including CEOs, take high level, short-term but long-lasting and far-reaching decisions also regarding the focus and fate of R&D, their qualification should also be high-level regarding understanding science and regulatory issues involved. In effect, corporate strategy leaders should actively engage in research and development, which also requires a deep understanding of the relevant scientific background, and the problems of risk-based decisions–before and after study results become available. In their own interest stakeholders and investors in the R&D based pharmaceutical industry should make sure that only those executive managers will lead the companies and make decisions on R&D that have proven to be highly qualified also in this specific sense. | Other | other | en | 0.999996 |
PMC11695962 | Section title: Introduction Educational score: 3.948643922805786 Domain: biomedical Document type: Other Language: en Cabotegravir (CAB) and rilpivirine (RPV) long-acting (LA) have become the first complete non-oral antiretroviral therapy (ART) regimen, approved for routine use in people with HIV (PWH) without resistance toward non-nucleoside/nucleotide reverse transcriptase (NNRTI) or integrase inhibitors (INI) or a history of virologic failure (VF) under an INI- and/or NNRTI-containing regimen. Section title: Introduction Educational score: 3.8689374923706055 Domain: biomedical Document type: Study Language: en While for many, CAB and RPV LA is a promising regimen for PWH struggling with daily intake of ART for various reasons, it is also faced with considerable skepticism, particularly from healthcare providers. There is concern about the ability of PWH to hold appointments in a time frame of ±7 days around the due day of the injection , in order to avoid sub-therapeutic drug concentrations that could favor the development of resistance. Adding to this concern, the development of VF with and without therapy-emerging resistance has been observed in the clinical trial program . Another specific concern around injected drugs is the occurrence of injection-site reactions (ISRs) that might contribute to high rates of discontinuation. Section title: Introduction Educational score: 4.058302402496338 Domain: biomedical Document type: Study Language: en This study aimed to estimate the probability of VF in particular, as well as discontinuation for any reason in general based on data from two-monthly (Q2M) CAB and RPV LA in a real-world cohort, incorporating prior findings from the Q2M arm of the ATLAS-2M, as well as the SOLAR trials , using Bayesian methodology. Section title: Materials and methods Educational score: 1.3672783374786377 Domain: biomedical Document type: Other Language: da Study design Section title: Materials and methods Educational score: 2.213291883468628 Domain: biomedical Document type: Study Language: en A retrospective, single-center, longitudinal, observational study in a large outpatient HIV research and clinical care center (Medizinisches Versorgungszentrum München am Goetheplatz) in Munich, Germany. The use of anonymized clinical routine data from a single center did not require the Ethics Committee’s approval. Section title: Materials and methods Educational score: 2.023653507232666 Domain: biomedical Document type: Study Language: en Inclusion and exclusion criteria Section title: Materials and methods Educational score: 3.337709665298462 Domain: biomedical Document type: Study Language: en The inclusion criterion was having received at least one dose of CAB and RPV LA after December 2020; people being on CAB and RPV for less than 48 weeks and who had not experienced VF or discontinuation for any reason, people participating in clinical trials, or who never received at least one dose of CAB and RPV LA were excluded from the analysis. Section title: Materials and methods Educational score: 2.0844736099243164 Domain: biomedical Document type: Study Language: en Objectives and outcome measures Section title: Materials and methods Educational score: 3.9985382556915283 Domain: biomedical Document type: Study Language: en The primary outcome measure was VF (two consecutive plasma HIV1-RNA concentrations >200 copies/mL and/or discontinuation due to virologic inefficacy). Primary and secondary objectives were the estimation of the probabilities of VF and discontinuation for any reason at week 48 (W48) after CAB and RPV LA initiation, respectively. Section title: Materials and methods Educational score: 1.2392867803573608 Domain: biomedical Document type: Other Language: en Statistics Section title: Materials and methods Educational score: 4.145367622375488 Domain: biomedical Document type: Study Language: en Using Bayesian methodology, the likelihoods of VF and discontinuation in the study were assumed to follow binomial distributions, with the risk set containing all PWH who had a follow-up of at least 48 weeks after initiation or discontinued before W48 (for reasons of comparability to the results from the snapshot analyses on the intention-to-treat-exposed (ITT-E), which were used for the construction of the prior). Conjugate beta priors were derived from the combined W48 results of the Q2M arm in ALTAS-2M , as well as the SOLAR trial with the ITT-E population as the risk set for VF and discontinuation at W48, respectively. The beta-binomial posterior distributions were approximated using Markov-Chain Monte Carlo methods with 5,000 iterations and a burn-in period of 1,000 for each of 10 chains. Mixing was investigated visually using trace plots while the stationarity of the chains was tested using Gelman-Rubin’s diagnostic for multiple chains. More details on the methodology can be found in the Appendix. Sensitivity analyses were performed using priors from ATLAS-2M (pessimistic approach) as well as SOLAR (optimistic approach) only (e.g. without combining the findings). Section title: Materials and methods Educational score: 2.4919321537017822 Domain: biomedical Document type: Study Language: en All analyses were performed using R 4.2.0 (R Foundation for Statistical Computing, Vienna, Austria). For all analyses, parameter estimates together with 95% highest posterior density credibility intervals were calculated. Sensitivity analyses were performed by using both, an optimistic as well as a pessimistic prior based on the published data. Section title: Results Educational score: 3.9902093410491943 Domain: biomedical Document type: Study Language: en Overall, 237 PWH were prescribed CAB and RPV between December 2020 and October 2023; 175 remained after applying inclusion and exclusion criteria and were used as the risk set for the primary analysis (Table 1 ). Most PWH initiating CAB and RPV had HIV-1 RNA <50 copies/mL; the highest HIV-1 RNA at first injection was found to be 82 copies/mL. Section title: Results Educational score: 4.109737396240234 Domain: biomedical Document type: Study Language: en Based on the results from the Q2M arm of ATLAS-2M and SOLAR trials, a β (12, 973) distribution was derived as the prior (details can be found in the Appendix). In our study sample, two PWH (1.1%) met the criteria of VF through W48 (occurring at months 5 and 9, respectively), resulting in a binomial (175, 0.011) likelihood, leading to a posterior probability of VL at W48 of 1.2% [0.6%; 1.9%]. Section title: Results Educational score: 4.002978324890137 Domain: biomedical Document type: Study Language: en For the secondary objective, i.e. discontinuation for any reason through W48, three PWH were excluded, who had planned to have a one-time CAB and RPV LA application only. Based on the reported discontinuation, a β (71, 910) distribution was derived as the prior (details can be found in the Appendix). In our study, 37 (21.4%) discontinued by W48, resulting in a binomial (172, 0.214) likelihood, leading to a posterior probability of discontinuation at W48 of 8.9% [7.3%; 10.5%]. Section title: Results Educational score: 4.067964553833008 Domain: biomedical Document type: Study Language: en Reasons for discontinuation are displayed in Table 2 . Among the local side effects, there was one abscess that led to discontinuation, while all other ISRs were pain with or without swelling and reddening and in general not severe (n=10). Among the systemic side effects, there was one immediate reaction to the injection that was considered either allergic or due to inadvertent intravascular injection. Other systemic side effects were headaches (n=1), worsening of a depressive episode (n=2), fatigue (n=1), impaired liver function tests (n=1), as well as pyrexia (n=1). Two discontinuations were due to suspected adverse events without further information. Preemptive discontinuation included discontinuation for previously unnoticed proviral resistance with a possible effect on RPV and/or CAB (n=6), problems in adherence (n=1), and long-time unavailability of injections (n=2). The recorded death was not ART-related and occurred in a state of advanced malignant disease. Section title: Results Educational score: 4.021537780761719 Domain: biomedical Document type: Study Language: en For a Bayesian sensitivity analysis, we varied the prior assumptions as follows: For a conservative approach to estimating the rate of VF, the prior was only based on the Q2M arm of the ATLAS-2M trial, which led to a posterior probability of VF of 1.6% [0.7%; 2.5%] while basing the analysis on the prior information from SOLAR only, it resulted in 1.0% [0.3%, 1.7%]. Section title: Discussion Educational score: 4.158547878265381 Domain: biomedical Document type: Study Language: en Our findings support the notion of VF being a rare event under CAB and RPV LA Q2M even outside clinical trials with two out of 175 PWH (1.1%) in the study sample fulfilling the criteria at W48. As estimating an incidence of rare events is generally imprecise, Bayesian methodology lends itself to estimating a probability of VF, taking previous information from clinical trials and our real-world findings into account. Combining the pooled prior knowledge with our data in a Bayesian manner, an updated probability of VF of 1.2% [0.6; 1.9] was estimated. This implies that VF does not occur more often in clinical routine than in clinical trials. During the 2024 Conference on Retroviruses and Opportunistic Infections and in medical journals, several data were published, reporting rates of VF under CAB/RPV LA in real-life settings as well as a clinical trial in Africa of 0.4-4.0% . These findings might be highly relevant in decision-making for CAB and RPV LA, as a frequent concern particularly among healthcare professionals might be the fear of a (much) higher rate of VF in the real world and lead to the fact that fewer people are given the possibility to profit from LA as an alternative to oral ART. Section title: Discussion Educational score: 4.0336408615112305 Domain: biomedical Document type: Study Language: en For discontinuation for any reason, rates of 6.9% and 9.5% were reported in ATLAS-2M and SOLAR through W48, respectively, while we observed a discontinuation rate as high as 21.4%. The discrepancy is, however, probably not surprising and can very likely be related to a stricter selection of PWH that enter clinical trials. Offering the possibility to experience CAB and RPV LA in clinical routine more widely to PWH who demonstrate interest, probably despite some doubts, will therefore very likely lead to a higher rate of discontinuation. We do not see the increased rate of discontinuation as a sign of an adverse safety profile in clinical routine; the degree of ISRs that contributed most to the discontinuations was not more severe than in the center’s long-standing experience in clinical trials but led more often to discontinuation. Section title: Discussion Educational score: 4.1253437995910645 Domain: biomedical Document type: Study Language: en Our study has several limitations. Despite having a considerable number of PWH on CAB and RPV LA included in this analysis, the sample size is still small compared to the ATLAS-2M and SOLAR trials. Therefore, the impact of the real-world findings is dominated by the prior, which in particular for the question on discontinuation might be relevant, as prior and study data lead to markedly different results and are therefore in conflict to some extent. Furthermore, being a single-center experience, our data may not reflect the heterogeneity of real-world conditions throughout different centers. In particular, being a center with early implementation and a very patient-centered approach to medical care with a liberal attitude toward innovations, a higher rate of discontinuation might have been expected with a higher number of PWH being given the opportunity to experience CAB and RPV LA even in cases where people might have been unsure. Also, it must be kept in mind that many people were taken off CAB and RPV preemptively due to previously unknown findings (including pre-existing resistance-associated mutations in proviral resistance tests); one might (rightfully) argue that these people might not have been started on CAB and RPV LA in the first place and that these PWH contributed relevantly to the excess probability of discontinuation. Further research might use bigger cohorts with a wider variety of PWH but also clinical settings to obtain more robust estimates of both VF as well as discontinuation for any reason. Section title: Conclusions Educational score: 4.167843818664551 Domain: biomedical Document type: Study Language: en Our data demonstrate that the rate of VF in the real world does not seem to exceed the expectations derived from the results of clinical trials, despite a less-controlled setting and a more diverse population given the opportunity to experience CAB and RPV LA. While there might therefore be higher rates of discontinuation, they are not driven by high rates of VF or severe adverse events. The most frequent reasons for discontinuation were ISRs and patient decisions, which could be seen as the result of balanced, informed decisions of PWH, in whom the benefit of LA therapies does not outweigh their downsides; this remains, however, speculative. On the other hand, several preemptive discontinuations (for example, due to medical details that only got available after the switch, such as resistance tests) might have led to a higher rate of discontinuation in real life than in clinical trials; in the setting of a clinical trial, these patients would very likely not have entered the trial in the first place. A more detailed comparison between the real-world data and the clinical trial data could be used to investigate whether the "threshold for discontinuation" is lower in real life than in the clinical trials or if the safety profile of CAB and RPV LA is adverse in clinical routine. While the analyses are dominated by the prior probabilities due to the high number of PWH included in the underlying trials, it seems recommendable to collect further real-world data from a broader range of PWH from multicenter cohorts to obtain a more stable estimate for both efficacy and safety of CAB and RPV in clinical routine. | Other | biomedical | en | 0.999997 |
PMC11696150 | Section title: Introduction Educational score: 2.1328208446502686 Domain: biomedical Document type: Other Language: en With more than one billion people, or approximately 1 in 7 individuals, living with some form of disability globally, and over 100 million children among them, the scale of this issue underscores the importance of addressing accessibility, inclusion, and support for individuals with disabilities across all sectors of society. These numbers highlight the significant impact disabilities have on individuals and communities worldwide, emphasizing the need for continued efforts to promote equality, empowerment, and opportunities for people of all abilities. It’s crucial to recognize that disabilities come in various forms, ranging from physical and sensory impairments to intellectual and developmental challenges, each requiring tailored support and accommodations to ensure full participation and inclusion in society . Section title: Introduction Educational score: 1.6533067226409912 Domain: biomedical Document type: Other Language: en The prevalence of disabilities in the Kingdom of Saudi Arabia accounts for 7.1% of the total population. According to the General Authority for Statistics, there are approximately 1,445,723 individuals with disabilities out of a total of 32 million people. This comprises 52.2% of males and 47.8% of females. Among the disabled population, an estimated 102,865 individuals have multiple disabilities . Section title: Introduction Educational score: 3.453784465789795 Domain: biomedical Document type: Study Language: en Notably, individuals with intellectual disabilities and autism require particular attention. Research indicates that 40% of individuals with intellectual disabilities also experience developmental disorders. Furthermore, around 30% of individuals with autism spectrum disorder (ASD) are also diagnosed with intellectual disabilities. Additionally, many experience co-occurring developmental disorders, such as attention deficit hyperactivity disorder (ADHD) or communication challenges . studies suggest that individuals with intellectual disabilities and autism often exhibit delays in verbal and linguistic behaviors . Section title: Introduction Educational score: 1.4445867538452148 Domain: other Document type: Other Language: en Skinner’s theory asserts that obstacles related to language and communication take specific forms based on the prevailing conditions within the verbal community . Consequently, these obstacles necessitate assistance from others. When a child makes a specific request, the reinforcement of that request becomes a motivating factor for the child. Requests hold great significance as they serve as the means through which speakers express their desires. The use of requests is strongly reinforced when the speaker’s request yields the desired results, often through unconditional reinforcement . Section title: Introduction Educational score: 1.7274794578552246 Domain: other Document type: Other Language: en The study conducted by Mukhopadhyay and Nwaogu highlights the significant challenge involved in teaching non-verbal students with intellectual disabilities. It was observed that the utilization of augmentative and alternative communication systems was not widespread in Botswana. Several studies, including those by Chambers and Rehfeldt , Curtis , Van der Meer et al. , and Achmadi have contributed valuable insights through literature review and theoretical frameworks. Section title: Introduction Educational score: 3.273846387863159 Domain: biomedical Document type: Other Language: en Augmentative devices refer to specialized tools or equipment designed to enhance the communication abilities of individuals with speech or language impairments. These devices form a key component of Augmentative and Alternative Communication (AAC) systems, encompassing both low-tech solutions, such as communication boards and picture-based systems, and advanced high-tech devices, including speech-generating devices (SGDs). Their primary purpose is to complement or replace existing communication methods, thereby enabling users to effectively express their thoughts, needs, and emotions across diverse settings . Section title: Introduction Educational score: 1.872336745262146 Domain: biomedical Document type: Other Language: en Individuals with multiple disabilities and communication disorders necessitate alternative communication methods. Moreover, children with communication disorders can effectively employ the PECS . Researchers in studies by Conklin and Mayer , Strogonova and Piper , and Al-Hamdi and Al-Farani have affirmed that individuals with multiple disabilities can be trained to use the PECS. Section title: Introduction Educational score: 2.7103030681610107 Domain: other Document type: Study Language: en Chambers and Rehfeldt conducted a study to compare the effectiveness of manual sign language and the PECS in teaching request skills to adults with severe intellectual disabilities. Four adults were taught to request four desired items using each communication method. The results indicated consistent performance across all four areas using the picture exchange system for three out of the four participants. Among these three participants, two later demonstrated standard performance in requesting items using manual sign language. Section title: Introduction Educational score: 3.8523855209350586 Domain: biomedical Document type: Study Language: en In a study conducted by Curtis , the objective was to determine the most effective approach for training children with autism spectrum disorder and intellectual disabilities in alternative communication methods. The study compared the effectiveness of manual sign language and the PECS in teaching six specific requests and increasing vocal behavior. The participants consisted of four children, aged (3) to (8), who had intellectual disabilities, autism spectrum disorder, or both, and exhibited limited communication skills. The results indicated that three participants successfully mastered requesting stimuli using the picture exchange system, while one participant achieved proficiency in requesting using manual sign language. Section title: Introduction Educational score: 3.7011921405792236 Domain: biomedical Document type: Study Language: en In Achmadi’s study , the aim was to identify the optimal method among three alternative communication approaches augmentative devices, manual sign language, and the picture exchange system for developing request skills. The study sample comprised four children, aged (4 to 5), with developmental disabilities, three of whom had autism and one had severe intellectual disabilities. The Vineland Adaptive Behavior Scale was utilized to assess the level of impairment, and a separate trial training strategy was employed. The training sessions were conducted in natural play environments at both home and school. The results indicated that three participants mastered the request skill using all three methods, while one participant achieved proficiency solely with the picture exchange system. Section title: Introduction Educational score: 2.6702699661254883 Domain: biomedical Document type: Study Language: en In contrast, Agius and Vance conducted a study to compare the effectiveness of two communication methods, namely the picture exchange system and the Sounding Board application on the iPad, in developing requesting skills in children under the age of six with autism spectrum disorder. The study involved a sample of three children. The results indicated that both the picture exchange system and the iPad with the Sounding Board application showed promise as effective tools for training requesting skills in preschool-aged children with autism. Section title: Introduction Educational score: 3.55610990524292 Domain: biomedical Document type: Study Language: en Furthermore, Abu Delhom conducted a study to evaluate the effectiveness of the PECS in fostering communication skills in children diagnosed with autism spectrum disorder. The study included a sample of (20) children randomly divided into two groups: a control group of 10 children and an experimental group of (10) children. Post-measurement and a four-month follow-up revealed statistically significant differences in language communication skills between the experimental group, trained on the picture exchange system, and the control group. These results favored the experimental group, indicating the positive impact of the picture exchange system on communication skills. Section title: Introduction Educational score: 2.9660723209381104 Domain: biomedical Document type: Study Language: en Similarly, Ganz and Simpson conducted a study with the objective of examining the PECS and its effectiveness in expanding acquired vocabulary. The study sample consisted of three children diagnosed with autism spectrum disorder who shared similar characteristics. The participants were trained in various stages of the picture exchange system, including How We Communicate, Distance and Persistence, Discriminating Pictures, and Building Sentences. The results demonstrated a clear increase in vocabulary and a higher number of words used in a single sentence, supporting the effectiveness of the picture exchange system in achieving the objective of expanding vocabulary. Section title: Introduction Educational score: 3.813880205154419 Domain: biomedical Document type: Study Language: en In a study conducted by Awaisat , the objective was to determine the effectiveness of alternative and augmentative communication methods in enhancing receptive and expressive language skills among individuals with severe intellectual disabilities. The study compared two methods: the picture exchange system utilizing picture exchange communication and the gestural method using the Makaton program. The sample consisted of (20) participants. The results indicated significant improvements in both receptive and expressive language skills for the experimental group trained on the picture exchange system and the experimental group trained on the Makaton program. However, no statistically significant differences were observed between the picture exchange system and the Makaton gestural program when applied to the experimental group in terms of receptive and expressive language skills. Section title: Introduction Educational score: 3.432904005050659 Domain: biomedical Document type: Study Language: en In another study, Park et al. investigated the impact of training parents in implementing the picture exchange system on the independent functional communication of three children diagnosed with autism spectrum disorder. The parents were taught the stages of the picture exchange system, including “How We Communicate,” “Distance and Persistence,” and “Discriminating Pictures.” The results demonstrated that the three children were able to independently exchange pictures, expanding their communication to various partners. Additionally, there was a limited improvement in speech production. Section title: Introduction Educational score: 3.7280561923980713 Domain: biomedical Document type: Study Language: en Similarly, Conklin and Mayer conducted a study to evaluate the effects of training in the picture exchange system using an MBD on three adults with developmental disabilities and severe communication impairments. Despite variations in training duration, all participants exhibited increased independent initiations after training on the picture exchange system. The results indicated a functional relationship between teaching the picture exchange system and enhancing independence, with continued improvements in independent initiations even after initial training. The participants trained on the picture exchange system also demonstrated increased initiation of requesting behaviors and subsequent improvements in their independence. Section title: Introduction Educational score: 3.884650230407715 Domain: biomedical Document type: Study Language: en Boesch et al. conducted a single-case experimental study to compare the effectiveness of the PECS and the Speech Generating Device (SGD) in developing requesting skills for three children under the age of 12 with severe autism spectrum disorder. The study found that both communication interventions significantly increased the requesting behavior of the children, providing alternative means of communication. However, the children faced challenges in picture discrimination. Interestingly, there was no significant difference observed between the PECS and the SGD, indicating that both approaches are equally suitable for developing requesting skills. Section title: Introduction Educational score: 3.484072685241699 Domain: biomedical Document type: Study Language: en In a similar vein, Strogonova and Piper investigated the effectiveness of the PECS in fostering communication and vocabulary development among children with intellectual disabilities. The study involved four children, fourteen parents, and eighteen teachers from the Riga Special Boarding School in Latvia. Through educational observation, the researchers assessed the skills and abilities of the participants at the beginning of the first semester and determined that the PECS was the most appropriate system for the sample. Importantly, all students readily accepted the introduction of the PECS. In addition, parents confirmed the utility of alternative communication tools, which could be actively incorporated into their children’s activities. Notably, the use of the PECS also led to a reduction in aggressive behaviors and improved the children’s ability to express their needs and emotions. Section title: Introduction Educational score: 2.1785056591033936 Domain: biomedical Document type: Study Language: en The current study is a valuable contribution to the scientific literature on the PECS within our Arab country. Its significance lies in its ability to assist researchers and individuals interested in addressing communication difficulties among children with multiple disabilities. Additionally, the study offers a theoretical contribution through the utilization of the MBD as an SSD. The study specifically focuses on children with multiple disabilities as participants. Section title: Introduction Educational score: 1.832138180732727 Domain: other Document type: Study Language: en During the researcher’s presence in daycare centers, they observed communication challenges among children with multiple disabilities. These challenges were attributed to the lack of specialized programs and services that prioritize the development of communication skills, particularly in the area of requesting. Motivated by these observations, the researcher decided to delve into this topic. The primary question addressed in this study is: What is the effectiveness of the PECS in developing requesting skills among children with multiple disabilities? Section title: Objectives Educational score: 1.9600023031234741 Domain: biomedical Document type: Study Language: en The study aims to achieve the following objectives: Section title: Method Educational score: 2.552788257598877 Domain: biomedical Document type: Study Language: en In this study, a single subject design was chosen as the most appropriate method to address the research objectives and interpret the study variables accurately. The specific experimental methodology employed in this study is the MBD. In essence, the implementation of the intervention during the study is expected to bring about changes in the targeted behavior. Section title: Design Educational score: 2.3152246475219727 Domain: biomedical Document type: Study Language: en The MBD was utilized by the researcher to assess the effectiveness of the PECS in developing requesting skills among children with multiple disabilities. Among various design options, the Multiple Baseline Across Participants design was chosen. This design is particularly advantageous in its ability to accurately validate the presence of a functional relationship between the independent variable and the dependent variable . Section title: Sample Educational score: 2.596705198287964 Domain: biomedical Document type: Study Language: en The study included a sample of three children with multiple disabilities who were enrolled in the Iiclusive Education Program at the Autism Excellence Center. The sample was purposefully selected and consisted of non-verbal children without hearing impairments, with intelligence quotient (IQ) scores ranging from 40 to 44 on the Stanford-Binet test. The participants’ chronological age ranged from 4.5 to 6.5 years. The specified criteria were carefully verified using a sample specification verification form, ensuring the accurate selection of the participating children (see Table 1 ). Section title: Targeted behavior and training program using PECS Educational score: 2.859257459640503 Domain: biomedical Document type: Study Language: en The current study provided a procedural description of the targeted behavior, which was identified as the requesting behavior, specifically requesting needs and reinforcements using the PECS. This behavior was selected based on reports from speech and language specialists and regular reports from the Autism Excellence Center, indicating expressive language difficulties and the inability of these children to make requests. The fourth phase of the PECS was utilized to develop the requesting skill. Section title: Targeted behavior and training program using PECS Educational score: 1.8085969686508179 Domain: biomedical Document type: Other Language: en The general objective of the program is to verify the effectiveness of using the PECS in developing the requesting skill for individuals with multiple disabilities. Section title: Targeted behavior and training program using PECS Educational score: 1.1374884843826294 Domain: other Document type: Other Language: en The training program was built upon several foundations, including: Section title: Targeted behavior and training program using PECS Educational score: 1.3566821813583374 Domain: other Document type: Other Language: en The program encompasses the following key components: Section title: Targeted behavior and training program using PECS Educational score: 1.6709543466567993 Domain: other Document type: Other Language: en Following the initial development of the training program, it underwent a comprehensive review by a panel of experts who provided valuable insights and feedback. Incorporating their suggestions, the training program was refined and enhanced to ensure its efficacy. The training program’s implementation mechanism, which utilizes the PECS to develop requesting skills, is explained, as depicted in the following Table 2 : Section title: Study tools Educational score: 1.7679187059402466 Domain: biomedical Document type: Study Language: en In order to achieve the objectives of the study, the following tools were developed and utilized: Section title: Study tools Educational score: 2.0015671253204346 Domain: biomedical Document type: Study Language: en The researcher presented the study tools to a panel of experts, who provided their opinions and recommendations. Necessary modifications were made to the tools based on their guidance, ensuring their validity and reliability for the study. Section title: Reliability of study procedures Educational score: 3.7691495418548584 Domain: biomedical Document type: Study Language: en To ensure the reliability of the study procedures, an independent observer was appointed from the specialists within the Autism Excellence Center. This observer had the responsibility of conducting the observation process. Initially, the researcher verified the reliability of the entire session procedures by providing the independent observer with a meticulously prepared form. The form encompassed the specific application procedures implemented by the researcher during the training sessions with the participating children. These procedures comprised a total of (8) steps, which can be referred to in Appendix (10) for further details. The role of the independent observer was to mark “correct” if the step was executed and “incorrect” if it was not executed. However, an important condition was established: The observer’s markings should be in agreement with the researcher’s execution in (33%) of the total conducted sessions. The number of sessions corresponding to this percentage was determined using the following equation: Section title: Reliability of study procedures Educational score: 1.809653878211975 Domain: biomedical Document type: Study Language: en Total number of training sessions × 0.33. Section title: Reliability of study procedures Educational score: 1.8604044914245605 Domain: biomedical Document type: Study Language: en Based on this, the researcher conducted a total of (7) training sessions, which is equal to (33%) of the sessions. The independent observer applied the form in these sessions. This calculation was done as follows: Section title: Reliability of study procedures Educational score: 1.6627568006515503 Domain: biomedical Document type: Other Language: unknown 19 ∗ 0 , 33 = 6.27 = 7 Section title: Reliability of study procedures Educational score: 1.8465734720230103 Domain: biomedical Document type: Study Language: en The researcher also ensured the reliability of each session’s procedures using the following equation: Section title: Reliability of study procedures Educational score: 1.5903443098068237 Domain: other Document type: Other Language: en number of executed steps / total number of steps ∗ 100 Section title: Reliability of study procedures Educational score: 2.211162805557251 Domain: biomedical Document type: Study Language: en Thus, the percentage of reliability for implementing the PECS procedures for each participating child during a single session ranged between 87.5 and 100% over the course of seven training sessions for all children. Section title: Reliability of study procedures Educational score: 2.360649585723877 Domain: biomedical Document type: Study Language: en To calculate the overall average reliability of the study procedures, the following equation was used: Section title: Reliability of study procedures Educational score: 1.8905202150344849 Domain: biomedical Document type: Other Language: en sum of session reliability percentages / number of observed sessions ∗ 100 Section title: Reliability of study procedures Educational score: 2.410287380218506 Domain: biomedical Document type: Study Language: en Applying this equation, the overall average reliability for implementing the PECS procedures for all children across the observed sessions was calculated as follows: Section title: Reliability of study procedures Educational score: 1.2261651754379272 Domain: biomedical Document type: Other Language: unknown 687.5 / 7 = 98.21 % Section title: Reliability of study procedures Educational score: 2.1404616832733154 Domain: biomedical Document type: Study Language: en Therefore, the overall average value indicates a strong reliability in the researcher’s implementation of the PECS procedures, as depicted in the table for calculating the overall reliability percentage of the study procedures (see Table 3 ). Section title: Inter-rater reliability Educational score: 2.699129343032837 Domain: biomedical Document type: Study Language: en The researcher assessed the inter-rater reliability between the observers (researcher and independent observer) to ensure the accuracy of recording children’s responses for each participating child during their training sessions using the PECS to develop requesting skills. The reliability of recording children’s responses was measured during (33%) of the sessions for each child using a “Child Response Recording Form” during the individualized program implementation. It was ensured that both the researcher and the independent observer recorded independently, without intervention or influence. Section title: Inter-rater agreement percentage for recording children’s responses Educational score: 2.1647961139678955 Domain: biomedical Document type: Study Language: en Recording Responses for the Implementation of the PECS: According to the researcher, the inter-rater agreement percentage for recording children’s responses was calculated using the following equation: Section title: Inter-rater agreement percentage for recording children’s responses Educational score: 1.7649579048156738 Domain: biomedical Document type: Other Language: en number of agreements / number of agreements + number of disagreements ∗ 100 Section title: Inter-rater agreement percentage for recording children’s responses Educational score: 2.1355862617492676 Domain: biomedical Document type: Study Language: en Thus, the inter-rater agreement percentage for recording children’s responses to the implementation of the PECS procedures for each participating child ranged between 90 and 100% over the course of seven training sessions for all children. Section title: Inter-rater agreement percentage for recording children’s responses Educational score: 2.2641334533691406 Domain: biomedical Document type: Study Language: en To calculate the overall average of the inter-rater reliability for recording children’s responses in all observed sessions, the following equation was used: Section title: Inter-rater agreement percentage for recording children’s responses Educational score: 2.5618574619293213 Domain: biomedical Document type: Study Language: en sum of inter − rater agreement percentages / number of observed intervention sessions = average Section title: Inter-rater agreement percentage for recording children’s responses Educational score: 2.5442183017730713 Domain: biomedical Document type: Study Language: en Using this equation, the overall average inter-rater reliability for recording children’s responses to the implementation of the PECS procedures for all children and all observed sessions was calculated as follows: Section title: Inter-rater agreement percentage for recording children’s responses Educational score: 1.1376582384109497 Domain: biomedical Document type: Other Language: unknown 680 / 7 = 97.14 % Section title: Inter-rater agreement percentage for recording children’s responses Educational score: 2.013320207595825 Domain: biomedical Document type: Study Language: en Therefore, the overall average value indicates a good indicator for the researcher’s recording of the independent variable’s results and data, influencing the dependent variable in this study. Refer to Table 4 for more information. Section title: Validity of study procedures Educational score: 2.087949514389038 Domain: biomedical Document type: Study Language: en The researcher rigorously established the validity of the study procedures, ensuring that the utilization of the PECS was indeed responsible for the observed changes in children’s behavior related to requesting. This was accomplished through the following meticulous steps: Section title: Statistical methods used Educational score: 3.6781418323516846 Domain: biomedical Document type: Study Language: en In this study, the researcher applied the MBD as an SSD, and utilized several statistical methods to analyze the data and derive meaningful results. These methods included reading frequency tables, calculating means, determining percentages of sample individuals, and conducting visual analysis of graphs . The purpose of employing these statistical techniques was to assess the effectiveness of the PECS in enhancing the requesting skill among children with multiple disabilities. Additionally, the performance of each student in the study sample was compared before and after the implementation of the study procedures. Section title: Study results and discussion Educational score: 2.0532443523406982 Domain: biomedical Document type: Study Language: en The first research question: What is the effectiveness of the PECS in developing requesting skills among children with multiple disabilities? Section title: Study results and discussion Educational score: 1.9229117631912231 Domain: biomedical Document type: Study Language: en To address this research question, the researcher presented the results obtained from children with multiple disabilities as they demonstrated an increase in requesting behavior through the utilization of the PECS. The results can be summarized as follows: Section title: Bader Educational score: 2.218440532684326 Domain: biomedical Document type: Other Language: en The child “Bader” demonstrated significant improvement in his requesting behavior, achieving the predetermined criterion of 100% on the graph over three consecutive sessions. This progress was observed during seven intervention sessions utilizing the PECS. Prior to the baseline phase, the researcher confirmed that “Bader” had not received prior training on the fourth stage of the PECS. To verify the desired behavior and assess its visibility to the specialist, the researcher provided a form to the responsible specialist at the Autism Excellence Center. This step was crucial in determining the need for appropriate intervention to enhance this behavior. Section title: Bader Educational score: 1.943210244178772 Domain: other Document type: Study Language: en During the baseline phase, the researcher systematically measured the requesting behavior using a specific form, enabling a logical assessment of the presence of a functional relationship between the independent variable (Fahad’s System) and the observed changes in Bader’s requesting behavior. Section title: Fahad Educational score: 2.2635514736175537 Domain: biomedical Document type: Other Language: en The child “Fahad” demonstrated significant improvement in his requesting behavior, achieving the predetermined criterion of 100% on the graph over three consecutive sessions. This progress was observed during seven intervention sessions utilizing the PECS. Prior to the baseline phase, the researcher confirmed that “Fahad” had not received prior training on the fourth stage of the PECS. To verify the desired behavior and assess its visibility to the specialist, the researcher provided a form to the responsible specialist at the Autism Excellence Center. This step was crucial in determining the need for appropriate intervention to enhance this behavior. Section title: Fahad Educational score: 2.537240743637085 Domain: biomedical Document type: Study Language: en At the beginning of the baseline phase, the researcher systematically measured the requesting behavior by making observations and utilizing a specific form tailored for this purpose. This procedure was implemented to logically evaluate the existence of a functional relationship between the independent variable (the PECS) and the dependent variable, which is the requesting skill. Its aim was to examine whether the implementation of the PECS had an observable impact on the child’s ability to make requests. Section title: Meshari Educational score: 2.28953218460083 Domain: biomedical Document type: Other Language: en The child “Meshari” demonstrated significant improvement in his requesting behavior, achieving the predetermined criterion of (100%) on the graph over three consecutive sessions. This progress was observed during seven intervention sessions utilizing the PECS. Prior to the baseline phase, the researcher confirmed that “Meshari” had not received prior training on the fourth stage of the PECS. To verify the desired behavior and assess its visibility to the specialist, the researcher provided a form to the responsible specialist at the Autism Excellence Center. This step was crucial in determining the need for appropriate intervention to enhance this behavior. Section title: Meshari Educational score: 2.346400260925293 Domain: biomedical Document type: Study Language: en During the baseline phase, the researcher carefully assessed the requesting behavior by making observations and utilizing a specific form designed for this purpose. This procedure was implemented to logically evaluate the existence of a functional relationship between the independent variable (the PECS) and the dependent variable, which represents the requesting skill. Section title: Meshari Educational score: 3.1717231273651123 Domain: biomedical Document type: Study Language: en The previous results, summarized in Figure 1 and Table 5 , clearly demonstrated a notable improvement in requesting behavior when children utilized the PECS. Remarkably, all children successfully met the predetermined criterion of achieving 100% on the requesting behavior graph for three consecutive sessions. The acquisition of this skill occurred through a series of intervention sessions, with each child requiring between five to seven sessions out of a total of 28 sessions, including maintenance and generalization sessions, as indicated in Table 6 . Section title: Discussion of results Educational score: 2.2363786697387695 Domain: other Document type: Study Language: en The results obtained from all the children demonstrate positive outcomes and highlight their proficiency in utilizing the PECS to enhance their requesting skills. These results provide solid evidence for the effectiveness of the PECS in promoting the development of requesting abilities. Moreover, they underscore the robustness of the implemented training program, which was meticulously designed by the researcher to cater to the individual needs and preferences of the children. The program’s success can be attributed to the careful consideration of their preferred reinforcers and motivators. Section title: Discussion of results Educational score: 2.1633989810943604 Domain: biomedical Document type: Study Language: en While variations in performance and differences among the children were observed, these can be attributed to their individual abilities and capabilities. However, thanks to the careful selection and homogeneity of the sample, as well as the development of a structured training program tailored to the abilities of all the children, they were able to achieve the predefined criterion. This criterion entailed reaching a consistent 100% level of requesting behavior on the graph during three consecutive sessions. Section title: Discussion of results Educational score: 3.8237314224243164 Domain: biomedical Document type: Study Language: en Similarly, Van der Meer et al. conducted a study involving four participants under the age of 18 with intellectual disabilities or autism, who had limited verbal abilities and adequate hand motor skills for request training. The participants underwent a five-day training program, consisting of four daily sessions. The results demonstrated that all four participants achieved mastery in requesting their preferred items using both the picture exchange system and augmentative devices, whereas only two of them acquired the skill of requesting through manual sign language. Section title: Discussion of results Educational score: 3.368809223175049 Domain: biomedical Document type: Study Language: en For example, both “Bader” and his peer “Fahad” replicated the requesting behavior using the PECS within the same time frame of seven days, spanning seven sessions. The similarity in the number of sessions can be attributed to the children’s concerted efforts to grasp the application mechanism effectively, coupled with their overall skill acquisition level. It is worth noting that during the intervention phase, Bader encountered a clear plateau in Session 4, where no progress was observed. As previously mentioned, this can be attributed to Badr having the least developed skill acquisition among the three children. However, after the fourth intervention session, Bader met the criterion and maintained this achievement throughout the subsequent maintenance phase, which commenced in Session 4. To clarify the methodology, after the last intervention session (Session 11), a waiting period of three sessions was implemented, and data for the maintenance session were collected in the fourth session. This approach was consistently followed for all maintenance sessions, including Sessions 23, 24, and 25. These sessions were conducted four days apart, and since no interventions occurred during these sessions, it becomes challenging to distinguish them in terms of behavior maintenance across all children. Section title: Discussion of results Educational score: 1.818773627281189 Domain: other Document type: Other Language: en Regarding the second child, “Fahad,” he successfully replicated the requesting behavior using the PECS within a consistent timeframe of seven days, spanning seven sessions, similar to his peer “Bader” as previously mentioned. Fahad achieved the predefined criterion during Session 4 of the intervention, and the same maintenance procedures were applied as with Bader. Section title: Discussion of results Educational score: 2.4284746646881104 Domain: biomedical Document type: Study Language: en Furthermore, the child “Meshari” demonstrated even faster progress, replicating the requesting behavior using the PECS within a shorter period of five days, encompassing five sessions. Meshari surpassed both Bader and Fahad in this regard. This exceptional performance can be attributed to Meshari’s enhanced ability to comprehend the application mechanism and overall skill acquisition. Notably, Meshari achieved the predefined criterion in the third session of the intervention, and the same maintenance procedures were followed as with Bader and Fahad. Section title: Discussion of results Educational score: 2.2031219005584717 Domain: biomedical Document type: Study Language: en The researcher suggests that the convergence among the children in terms of the number of sessions required during the intervention can be attributed to several factors, including intelligence level, mental age, and chronological age. Additionally, the researcher proposes that the convergence in the development of requesting skills using the PECS can be attributed to the remarkable ability of children with multiple disabilities to comprehend the system, despite their young age. Section title: Discussion of results Educational score: 1.99514901638031 Domain: biomedical Document type: Other Language: en Three primary factors contribute to the children’s progress: joint assessment, reinforcement evaluation, and the number of training attempts before the intervention data collection session. All children were provided with a significant number of well-structured educational opportunities, facilitating the development of the targeted behavior. These factors directly contributed to the children with multiple disabilities’ ability to comprehend the training program’s defined steps and procedures. Section title: Discussion of results Educational score: 2.7849671840667725 Domain: other Document type: Study Language: en The obtained results are in line with the results of Abu Delhom , which emphasize the role of the PECS in developing communication skills. Similarly, they align with Awaisat’s study , which observed improvements in expressive language skills through the implementation of a picture exchange system. Consistent with Conklin and Mayer , Boesch et al. , and Strogonova and Piper , this study demonstrates that participants who underwent training with the PECS experienced enhancements in their requesting abilities. Section title: Conclusion Educational score: 2.5685136318206787 Domain: biomedical Document type: Study Language: en The present study corroborates the results of , supporting the notion that the utilization of the PECS leads to more effective communication skills. Furthermore, it aligns with Chambers and Rehfeldt , Curtis , and Van der Meer et al. emphasizing that children with multiple disabilities can successfully employ the PECS. Section title: Conclusion Educational score: 2.7537472248077393 Domain: biomedical Document type: Study Language: en This study consisted of five main chapters. The first chapter covered the introduction, research problem, and the study questions. It also presented the study objectives, which aimed to investigate the effectiveness of using the PECS in developing and maintaining the requesting skills of children with multiple disabilities, as well as generalizing these skills. The study objectives also included measuring the effectiveness of training children with multiple disabilities to use the PECS for developing requesting skills. The chapter emphasized the theoretical and practical significance of the study, outlined its limitations, and defined relevant terms. Section title: Conclusion Educational score: 2.04923415184021 Domain: biomedical Document type: Other Language: en In the second chapter, the researcher discussed the literature review, focusing on the following dimensions: Multiple disabilities. Intellectual disabilities. Autism spectrum disorder. SSDs (MBDs). The chapter also reviewed previous studies, which were divided into two dimensions: Reviewing previous studies that addressed requesting skills. Reviewing previous studies that addressed the use of the PECS. Section title: Conclusion Educational score: 3.367535352706909 Domain: biomedical Document type: Study Language: en The third chapter outlined the methodology and procedures of the study. It included the study’s approach and its population, the variables and sample characteristics, the sample selection procedures, descriptions of the participants, the study’s tools and their application procedures, the validity and reliability of the study procedures and the statistical methods used in the study. Section title: Conclusion Educational score: 1.7576860189437866 Domain: biomedical Document type: Other Language: en In the fourth chapter, the study results were presented, analyzed, and interpreted. This chapter included: analysis of the study’s instruments’ results and interpreting the results to answer the research questions. Section title: Conclusion Educational score: 1.7963968515396118 Domain: biomedical Document type: Other Language: en The fifth chapter covered the study’s summary. It included the conclusion, the key results, the recommendations, suggestions for future research and limitations of the current study. Section title: Recommendations Educational score: 1.8878508806228638 Domain: biomedical Document type: Other Language: en Based on the results of the current study, the following precise recommendations are suggested: Section title: Suggestions for further research Educational score: 1.4588643312454224 Domain: biomedical Document type: Other Language: en Several suggestions can be proposed to conduct future studies that build upon the results of the current study. These suggestions include: Section title: Study limitations Educational score: 1.9884096384048462 Domain: biomedical Document type: Study Language: en – Limited sample size targeted in the current study due to the utilization of SSDs, which, according to theoretical literature, recommend a small sample size . | Other | biomedical | en | 0.999995 |
PMC11696152 | Section title: 1 Introduction Educational score: 4.117647171020508 Domain: biomedical Document type: Review Language: en The prevalence of myopia continues to increase, such that it is expected to affect nearly half the global population by 2050 . Uncorrected myopia is one of the most frequent causes of visual impairment , and its progression can damage the retina, choroid and sclera . Although myopia is a major public health problem, how it occurs and how it involves different populations of cells in the eye are unclear. Various processes have been proposed to contribute to myopia, such as reduced dopamine signaling , choroidal thinning and associated ischemia , hypoxia and remodeling of the sclera , as well as inflammation and oxidative stress . The retina, as the first part of the eye that encounters abnormal visual signals, may play a central role in myopia by triggering excessive eye growth in response to those signals . Section title: 1 Introduction Educational score: 3.8266422748565674 Domain: biomedical Document type: Review Language: en Retinal glia, which outnumber retinal neurons by at least a factor of 10 , are likely to be involved in myopia. Here we review our current understanding of how different populations of glia in the retina may be involved in myopia, and we propose future directions for research to deepen that understanding. Section title: 2 Concise anatomy of the retina and description of myopia Educational score: 4.38338041305542 Domain: biomedical Document type: Other Language: en The retina is a sophisticated visual sensory tissue with a laminar structure comprising the following ten layers from outside to inside : retinal pigmented epithelium (RPE) monolayer, photoreceptor layer (PL), outer limiting membrane (OLM); outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL), retinal nerve fiber layer (RNFL), and inner limiting membrane (ILM). Incoming light is sensed by photoreceptors in the ONL, which transmit visual signals directly and via bipolar cells in the INL. Horizontal cells and amacrine cells in the INL fine-tune the visual signal horizontally in the OPL and IPL, respectively. The fine-tuned signals are coordinated in retinal ganglion cells, which send the input to higher visual processing centers in the brain . Section title: 2 Concise anatomy of the retina and description of myopia Educational score: 4.459560394287109 Domain: biomedical Document type: Review Language: en Axial myopia involves substantial lengthening of the ocular axis, enlargement of the vitreous, reduced choroidal blood flow, and decreased choroidal thickness. The inner retina becomes thinner, especially the RNFL and IPL, than all the retinal layers . Although the onset of myopia remains unclear, ischemia-hypoxia, inflammation, and oxidative stress are recognized the potential pathological responses in myopia . Glia is implicated in those stress. Astrogliosis in myopia may influence blood oxygen supply, neuronal function, and axon diameter . Astrocytes and Müller cells provide structural support in normal retina . In ocular elongation suffering from mechanical stress in myopia, Müller cells especially may act as a sensor of mechanical stretching . Their abnormalities lead to fragile structure of retina to cope with intraocular pressure and mechanical forces with fast-growing of eyeball. The downstream molecular responses within Müller cells can transmit glial signals and synchronize the activities of many other neurons . Müller cells play roles in retinal extracellular matrix remodeling, providing an incipient looser microenvironment for migration of cells, cytokines, or inflammatory factors in myopia . Microglia may exhibit a reactive morphology and elevated response to inflammation in myopia . Due to the limited evidence, it is hard to summary the role of glia in the onset of myopia, high myopia or pathological myopia respectively, or whether glia responses promote myopia progression. How each glial cell respond to abnormal visual experiences in myopia will be described in detail here. Section title: 3 Location, morphology, and function of glia in the retina Educational score: 4.063035488128662 Domain: biomedical Document type: Other Language: en Glia, comprising mainly microglia and two types of macroglia called astrocytes and Müller cells , reside primarily in the inner retina. Microglia sense inflammatory signals and strive to maintain homeostasis in the retina. Astrocytes modulate the activity of neurons and retinal ganglion cells in order to support the transfer of visual signals to higher vision centers in the brain. Müller cells respond to ocular stretching and ocular fluid regulation, thereby helping to regulate and refine visual signals to the outer ocular layers . Section title: 3.1 Astrocytes Educational score: 4.1458306312561035 Domain: biomedical Document type: Study Language: en Astrocytes migrate into the retina along with its vasculature and concentrate within the RNFL. The number and location of astrocytes in the retina strongly correlate with the number and location of blood vessels and nerve fibers there . The parafoveal and foveal regions feature four layers of vasculature and two layers of astrocytes, one of which is a superficial layer close to the ILM, while the other is a deeper layer close to the GCL. The peripapillary region contains three layers of vasculature and one layer of astrocytes. The peripheral region features two layers of vasculature and one layer of astrocytes . Section title: 3.1 Astrocytes Educational score: 4.166090965270996 Domain: biomedical Document type: Study Language: en The cell bodies of astrocytes aggregate in the RNFL, and their processes reach axons in the retinal ganglion cells as well as vasculature and other glia within the RNFL . Astrocytes can be identified based on their expression of glial fibrillary acidic protein (GFAP), the primary type of intermediate filament in the vertebrate nervous system . This and other intermediate filaments provide structural and mechanical support to maintain cellular morphology , while also coordinating mechanical sensing, transduction, signaling, motility, and inflammatory responses . Section title: 3.2 Müller cells Educational score: 4.198052883148193 Domain: biomedical Document type: Study Language: en From the somata of Müller cells in the INL radiate two stem processes in opposite directions, spanning from the ILM to the ONL, which is nearly the entire thickness of the retina. The inner stem process terminates in a funnel-shaped endfoot. Lateral processes extend into the plexiform layers to form sheaths around synapses, while also extending into the nuclear layers to embed in neuronal perikarya. Section title: 3.2 Müller cells Educational score: 4.513141632080078 Domain: biomedical Document type: Study Language: en Their unique morphology allows Müller cells to interact with all neurons of the retina and modulate synaptic activity . Müller cells play a crucial role in retinogenesis, transmit various molecules between different retinal cells and participate in the establishment and maturation of the blood–retinal barrier . They support neurons by releasing trophic factors and neurotransmitters as well as by regulating extracellular ion homeostasis. Like astrocytes, Müller cells can also be identified based on their expression of GFAP , and both types of macroglia provide mechanical support to the retina through strands of microtubules and intermediate filaments such as GFAP and vimentin . In addition to playing this supporting role in transmission of visual signals, Müller cells can themselves play the main role of light detection and phototransduction . Moreover, Müller cells contribute to the visual cycle of cone cells by phagocytosing their outer segments to promote their turnover and biosynthesis . Section title: 3.3 Microglia Educational score: 4.463043212890625 Domain: biomedical Document type: Study Language: en Microglia are the primary resident innate immune cells of the central nervous system. They contribute to programmed cell death, neurogenesis, vascular development, and refinement of synapses and neuronal circuits. In the healthy mature retina, microglia make up a stable and highly ordered network of ramified cells that are thought to carry out constitutive maintenance functions as well as regulate neuronal activity and synaptic integrity. They are usually distributed horizontally in the synaptic OPL, IPL and nuclear GCL of the retina, as well as around blood vessels . Microglia in the central nervous system derive from hematopoietic progenitors in the extraembryonic yolk sac . When microglia enter the developing retina, amoeboid cells that express markers of microglia or macrophages emerge in the vitreous and on the vitreal surface of the embryonic retina, near the optic nerve, and in the peripheral retina. Then they simultaneously proliferate and migrate radially and horizontally to occupy the entire retina. Their morphology becomes polarized and their processes ramify . Section title: 4 Myopia disproportionately affects the inner retina, where most glia cells localize Educational score: 4.304597854614258 Domain: biomedical Document type: Study Language: en Excessive eye growth in myopia leads to thinning that is more severe in the inner retina than in other retinal layers . Experiments in different systems suggest that the thinning affects the RNFL and IPL . Experiments in a marmoset model of myopic foveoschisis suggest that myopia involves not only mechanical stretching of the retina but also gliosis . In a marmoset model, myopia did not obviously affect photoreceptors in the outer retina . Electroretinographic studies of marmoset models support the idea that myopia affects primarily the inner retina and not the outer retina , and that changes of electrophysiology in bipolar, retinal ganglion, amacrine and glial cells may precede pathology of the inner retina . Section title: 4 Myopia disproportionately affects the inner retina, where most glia cells localize Educational score: 2.8905506134033203 Domain: biomedical Document type: Other Language: en The observation that myopia involves pathology primarily in the inner retina, where most glia localize, prompts the question, “How do retinal glia contribute to myopia?” Section title: 5.1 Astrogliosis Educational score: 4.627728462219238 Domain: biomedical Document type: Study Language: en Quiescent astrocytes are activated respond to adverse factors, leading to changes in morphology, gene expression, and functions, known as “astrogliosis” . One hallmark of astrogliosis is upregulation of GFAP. Such gliosis is a double-edged sword: it can protect retinal ganglion cells from further injury, yet it can also promote their death . Myopia is likely to involve astrogliosis . In a marmoset model of myopia, myopic eyes showed a smaller number of astrocytes yet higher expression of GFAP, consistent with astrogliosis . Astrocytes in the radial peripapillary capillary layer in fovea showed enlarged somata with thicker, shorter, and irregular processes . Astrocytes in the superficial vascular plexus in fovea, peripapillary, and peripheral retina also showed hypertrophy and hyperdense processes . These pathological changes worsened with time. Similar results were observed in a mouse model of myopia . The horizontal GFAP area in the myopic eye was thicker than in the control eye, and the myopic eye showed fibers deep in the IPL, which was much thicker than the control eye . In the control eye, GFAP expression was restricted to the inner side of retinal ganglion cells. In a rhesus macaque model of myopic foveoschisis, gliosis of macroglia was found at the central macula, where foveoschisis was most prominent and foveal pit morphology most severely disrupted, yet photoreceptors showed only minor disruption . Section title: 5.1 Astrogliosis Educational score: 4.204494476318359 Domain: biomedical Document type: Study Language: en These studies suggest that in myopia, astrocytes in foveal and peripheral retina show astrogliosis involving altered morphology and upregulation of GFAP. The formation of thin, long filaments running in one direction along the deeper vasculature of the RNFL, especially in peripapillary and peripheral retina, is probably the result of mechanical stretching. Myopia likely interacts with age to affect retinal astrocytes, given that aging is associated with decreased astrocyte density and astrogliosis . Section title: 5.1 Astrogliosis Educational score: 4.33419942855835 Domain: biomedical Document type: Study Language: en So far, direct evidence linking astrogliosis to myopia in humans is lacking, although glia cells have been detected in ILM from patients with myopic foveoschisis . The ILM is a specialized basement membrane located at the border between the vitreous body and retinal neuroepithelium. One possibility is that the ILM stiffens through upregulation of GFAP, reactive gliosis, abnormal collagen formation and long insertions of process of glia cells. On the other hand, such stiffening of the ILM can inhibit gliosis . Stiffening of the ILM may also generate severe traction force, putting significant mechanical stress onto foveal layers and causing foveoschisis lesions. Whatever the detailed mechanism(s), the development of myopia in humans involves transfer or reorganization of astrocytes , because ILM from myopic individuals shows increased astrocyte density . However, astrocyte density was decreased in all retinal regions of a marmoset model of myopia . Further research should explore the potential contribution of gliosis in myopia. Section title: 5.2 Astrogliosis and the astrocyte-neurovascular unit Educational score: 4.231905937194824 Domain: biomedical Document type: Study Language: en Every astrocyte in the retina contacts at least one blood vessel and at least one neuronal element such as the soma or axon of retinal ganglion cells, allowing them to guide vascular development and mediate the integration between blood vessels and neurons to create the so-called astrocyte-neurovascular unit . This unit appears to be crucial for retinal structure , modulation of vascular tone, regulation of blood flow and integrity of the blood-retinal barrier , as well as metabolism, neuronal turnover and neurotransmitter homeostasis . It seems reasonable to assume that astrogliosis in myopia can alter the physiology and processing activity of retinal ganglion cells and other neurons, leading to abnormal retinal vascularization and weakened structural support. Section title: 5.2 Astrogliosis and the astrocyte-neurovascular unit Educational score: 4.412419319152832 Domain: biomedical Document type: Study Language: en Consistent with this idea, myopic eyes in humans and animal models show decreased blood supply to the retina and simultaneous loss of astrocytes and their associated capillaries across the retina, together with slower blood flow in the central retinal artery , narrowing of retinal vessels , lower capillary density , larger avascular zones in the fovea , and loss of vascular branching in the periphery and peripapillary regions . Vascular branching in the fovea may increase as a compensatory mechanism, at least in a marmoset model of myopia . All these indicators of vascular reorganization suggest hypoxia in the myopic periphery . Supporting that, four genes were shared as reported between hypoxic astrocytes and human myopia, which are GRIA4 , RP2 , CNGB3 , and ADAMTS10 . GRIA4 is expressed in the cone ON bipolar cells and is responsible for the common refractive error. RP2 and CNGB3 are expressed in cones and rods and are associated with syndromic myopia. ADAMTS10 is expressed in the sclera . Section title: 5.2 Astrogliosis and the astrocyte-neurovascular unit Educational score: 4.464768886566162 Domain: biomedical Document type: Study Language: en In addition to affecting blood flow, astrogliosis in the astrocyte-neurovascular unit may also affect neuronal transmission. Astrogliosis has been linked to demyelination, compensatory remyelination, and axon loss in the developing and diseased brain . Normally, axons of retinal ganglion cells at the optic nerve head within the retina are unmyelinated, while the proportion of myelinated retinal ganglion cells increases as one moves toward the brain . In chickens, in which intraocular myelination of ganglion cell axons is normal, myopia reduced the thickness of the RNFL by about 14% and the thickness of unmyelinated axons by about 29%, while also reducing the total number of myelinated axons . The velocity and fidelity of visual signal conduction depend mainly on myelin sheath length and axon diameter : for example, larger axon diameter translates to faster signal conduction . Thus, demyelination and shrinking axon diameter may slow visual signal conduction and render the neurons more susceptible to hypoxic injury, especially in the presence of myopic pathology such as myopia-associated astrogliosis. Section title: 5.2 Astrogliosis and the astrocyte-neurovascular unit Educational score: 3.516099452972412 Domain: biomedical Document type: Other Language: en These considerations raise the question: what happens to unmyelinated retinal axons of humans or mice in the presence of myopia? Chronically abnormal visual stimuli may directly influence the axons without myelin, even though glia may play a compensatory role like myelin. Section title: 5.2 Astrogliosis and the astrocyte-neurovascular unit Educational score: 4.2208356857299805 Domain: biomedical Document type: Study Language: en Differential vulnerability of the astrocyte-neurovascular unit in different parts of the eye may help explain why the excessive growth in myopia disproportionately affects the peripheral retina. This region and the optic nerve head, which are less stiff than the mid-retina , contain astrocytes with compressed morphology that probably contact fewer unique blood vessels. As a result, astrocytes in these regions cannot regulate blood flow as effectively as in the mid-retina, especially in the presence of myopic injury . Section title: 6 Müller cells in myopia Educational score: 2.2709922790527344 Domain: biomedical Document type: Other Language: en The literature suggests numerous mechanisms through which Müller cells may contribute to myopia . Section title: 6.1 Müller cells and ATP production and degradation Educational score: 4.231310844421387 Domain: biomedical Document type: Study Language: en Inhibiting adenosine receptors, which are activated by the adenosine from ATP breakdown , inhibits myopia in animal models . The vitreous humor from myopic individuals with complications contains elevated level of uric acid , the end-product of ATP breakdown. These observations lead to the proposal that progression of myopia involve elevation in extracellular ATP and its degradation into adenosine and uric acid. This is consistent with the activity of extracellular ATP as a danger signal that can activate P2X or P2Y receptors to stimulate inflammation and other pathological signaling cascades . Section title: 6.1 Müller cells and ATP production and degradation Educational score: 4.30009651184082 Domain: biomedical Document type: Study Language: en Indirect evidence implicates Müller cells in the release of ATP outside cells in myopia. These cells can release ATP in response to light, osmotic or mechanical stress, as well as following activation of purinergic, dopaminergic and glutamatergic receptors . Activation of purinergic receptors has been shown to induce gliosis of Müller cells yet also their hypertrophy and proliferation . Exposing Müller cells to ATP, light, electrical or mechanical stimulation can trigger them to release Ca 2+ from intracellular stores, which synchronize the activities of many neurons and transmit glial signals across larger areas of the retina . Section title: 6.2 Müller cells and hypoxia Educational score: 4.249231815338135 Domain: biomedical Document type: Study Language: en A growing body of literature propose that hypoxia modulates myopia development . The longer axial length in myopia is associated with thinning of the choroids , which may in turn lead to less blood perfusion in retina. This creates a hypoxic environment for photoreceptors in the outer retina, which is one of the most metabolically demanding tissues and which relies mainly on perfusion of choroid tissue . Chronic hypoxia appears to upregulate secretion of basic fibroblast growth factor (bFGF) by Müller cells, which stimulates the proliferation of retinal vascular endothelial cells to drive neovascularization . Section title: 6.3 Müller cells and mechanical stretching Educational score: 4.4087958335876465 Domain: biomedical Document type: Study Language: en Myopia involves excessive elongation of the eye axis, which inevitably involves mechanical stretching. Müller cells can sense such stretching and other subtle alterations because of their unique transretinal morphology with long, branched processes. Stretching induces in Müller cells rapid, transient increases in intracellular Ca 2+ as well as slower, longer-lasting changes in gene expression , and it triggers responses involving transcriptional factors and molecules involved in ocular axial growth . Among these changes in expression is initial upregulation of bFGF , which in turn leads to upregulation of matrix metalloprotease (MMP)-2 , which cleaves proteins of the extracellular matrix and can make the retina less stiff, thereby protecting the retina at an early phase from damage induced by stretching. In the presence of chronic mechanical stress , levels of bFGF and MMP-2 decrease, which may also protect the eye by strengthening the retina and sclera from serious damage. While the initial upregulation and subsequent downregulation may serve to protect the eye, they may not so benefit for maintaining emmetropia. Section title: 6.4 Müller cells and remodeling of the extracellular matrix Educational score: 4.541406154632568 Domain: biomedical Document type: Study Language: en Müller cells may influence ocular growth not only by secreting MMPs that degrade the extracellular matrix (ECM), but also by secreting MMP inhibitors called tissue inhibitors of metalloproteases (TIMPs) . In fact, retinal damage induces Müller cells to upregulate their secretion of TIMP2 and downregulate their secretion of matrix-degrading gelatinase . In these ways, Müller cells may influence the balance between degradation and formation of the matrix , which in turn may loosen or stiffen the retina and alter its shape . Individuals with high myopia show upregulation of MMPs and TIMPs in the aqueous humor , while experiments in a rat model have suggested that knockout of TIMP4 can contribute to high myopia by reducing collagen content in the sclera and retina . Future research is needed to clarify in detail how the extracellular matrix in the retina is altered in myopia and what mechanisms drive those alterations. Such work should consider the apparently two-way communication between Müller cells and the extracellular matrix: the cells secrete MMPs and TIMPs to affect the matrix, while remodeling of the matrix influences the (de)differentiation and proliferation of Müller cells . Section title: 6.4 Müller cells and remodeling of the extracellular matrix Educational score: 4.103549003601074 Domain: biomedical Document type: Study Language: en Few attentions have been paid to how myopia involves extracellular matrix in the retina rather than in the sclera. The matrix is less abundant in the retina than the sclera, so responses of matrix deposition and remodeling in the retina may be less effective at resulting in ocular elongation. Instead, it prefers to providing incipient looser retinal microenvironment for the molecules, glia, and neurons to respond to abnormal visual experiences in myopia. Anomalous matrix deposition can stiffen the retina and trigger inflammation, leading to scar formation and fibrosis ; matrix degradation, conversely, can protect the retina from mechanical stress. Section title: 6.5 Müller cells and ILM remodeling Educational score: 4.454183578491211 Domain: biomedical Document type: Study Language: en The endfoot of Müller cells secretes serine protease 56 (PRSS56) into the ILM of the retina , and this protein, as well as the transmembrane glycoprotein membrane frizzled-related protein (MFRP), may help drive excessive growth of the ocular axis. Mutations in the genes encoding either protein lead to a shorter ocular axis in humans and mice , and loss of either protein reverses the ability of mutations in the gene encoding the interphotoreceptor retinoid-binding protein (IRBP) to drive excessive growth of the ocular axis . Given that MFRP is expressed predominantly in the retinal pigment epithelium and that IRBP is expressed primarily in the interphotoreceptor matrix between the retinal pigment epithelium and photoreceptors, these proteins, together with PRSS56, may mediate the ability of Müller cells to influence the retinal pigment epithelium and transmit information during ocular growth. Section title: 6.5 Müller cells and ILM remodeling Educational score: 3.793105125427246 Domain: biomedical Document type: Study Language: en The PRSS56 secreted by Müller cells may help degrade the extracellular matrix and remodel the ILM, which in turn may facilitate ocular axial growth . Providing mechanical support to the ILM or inner retina has been shown to reduce Müller cell gliosis and protect neurons in an in vitro model . These results, suggest a therapeutic strategy against myopia. Section title: 6.6 Müller cells and transretinal fluid movement Educational score: 4.227725505828857 Domain: biomedical Document type: Study Language: en Why the vitreous becomes enlarged in myopia remains a mystery, though numerous studies seem to suggest that the enlargement is the consequence of axial elongation rather than the passive result of increased vitreous or ocular volume . An unexplored possibility is that the enlargement occurs when abnormal visual experiences lead to neural activity that alters ion concentrations and osmotic potential, causing transretinal fluid movement into the vitreous. Müller cells are well-suited to facilitate transretinal fluid movement because of their morphology spanning the retina . Osmoregulation is crucial for the retina because its high energy demand and metabolic turnover require efficient systems for preventing water accumulation . Section title: 6.6 Müller cells and transretinal fluid movement Educational score: 4.758587837219238 Domain: biomedical Document type: Study Language: en The rate of net fluid transfer between the vitreous and choroid as well as alterations in choriocapillaris permeability may contribute to the redistribution of water in myopia. In a chicken model of myopia, rapid axial elongation and movement of fluid into the vitreous cavity were associated with upregulation of aquaporin 4 in the nerve fiber layer . In that model, upregulation of the inward-rectifying K + channel from the 4.1 subfamily (Kir4.1) appeared to limit axial elongation. These considerations are consistent with the central role of Müller cells in myopia, because the endfeet of these cells express aquaporin 4 at interfaces with retinal capillaries, the vitreoretinal border and synapses in the plexiform layers to facilitate retinal signal transduction . The endfeet of Müller cells facing the vitreous and blood vessels of the mammalian retina co-express aquaporin 4 and Kir4.1 , and these regions act as K + sinks to limit concentrations of K + in the extracellular space around active neurons . One possibility is that under normal conditions, aquaporin 4 in the inner retina supports rapid fluid flow across the retina into the vitreous, and it cooperates with ion cotransporters in the retinal pigment epithelium to transport fluid out of the retina and into the choroid. The abnormal visual signaling in myopia may disturb osmotic homeostasis and alter aquaporin 4 expression in Müller cells, leading to excess fluid movement and deposition in the vitreous chamber and, potentially, reduced fluid outflow into the choroid, ultimately leading to ocular enlargement . Consistent with this hypothesis, a chicken model of myopia showed substantial increases in levels of K + , Na + and Cl − in the outer retina as well as increases of Na + and Cl − in the inner retina . Section title: 6.6 Müller cells and transretinal fluid movement Educational score: 4.1914262771606445 Domain: biomedical Document type: Study Language: en One mechanism proposed for myopia is that as photoreceptors sense blurred images, the concentration of K + increases, and choroid vessels respond quickly to the increase in osmotic pressure in the retina, leading to excessive fluid accumulation. The endfeet of Müller cells in the outer retina sense the accumulation and attempt to compensate for it by upregulating aquaporin 4 and downregulating Kir4.1. The higher extracellular concentration of K + in myopia may lead to neuronal excitability, which should be explored in future research. Section title: 6.7 Müller cells and reprogramming Educational score: 4.282915115356445 Domain: biomedical Document type: Study Language: en Müller cells can dedifferentiate into progenitor cells and they can differentiate into a damaged cell type under the influence of cellular and environmental factors . Progenitors of Müller cells form a circumferential marginal zone (CMZ) that lines the periphery of the retina. Normally the retina provides signals that suppress proliferation of progenitors in the CMZ , but induction of myopia stimulates proliferation of those progenitors which is associated with eye growth . Glucagonergic amacrine cells with massive neurites cluster around the progenitors and may contribute to myopia progression . These new additional cells derived from proliferation of progenitors of Müller cells may not only enlarge retina, but also provide more abilities to differentiate into other functional neurons supporting the retina. Section title: 7 Microglia in myopia Educational score: 4.431628227233887 Domain: biomedical Document type: Study Language: en Quite little is known about the role of microglia in the progression of myopia . A single-cell RNA sequencing research performed on mouse discovered that microglia activity was increased in high myopic retinas . Il1a , Il6ra , Il21r (interleukin, IL) , Tgfbr1 , and Tgfbr2 (transforming growth factor-β, Tgfb) and downstream transcriptional regulators ( Stat3 , Nfkbr1 , and Nfkbr2 ) were found significantly increased in the microglia of highly myopic eyes. It indicates cytokine receptors rather than cytokines, and TGF-β receptors were significantly elevated in microglia which highlight the enhanced responses of highly myopic eyes to proinflammatory environment and the growth-promoting states involved in high myopia progression. STAT3 signaling pathway was activated in highly myopic microglia, exhibiting an aging or neuroinflammation profile. Meanwhile, genes enriched for cell activation, cell migration, and cellular responses to stress were also upregulated in highly myopic microglia . In animal models of myopia, activated microglia in the IPL showed shorter, thicker processes differing from the long, thin, highly branched processes in control retinas . In a primate model of pathologic myopic foveoschisis, activated microglia in the fovea showed amoeboid rather than normal dendritic morphology . Microglia in the peripheral retina, however, showed normal dendritic morphology, and photoreceptors in the retina did not show obvious alterations . Further research is needed to expand our knowledge of microglia in myopia. Section title: 8 Future directions Educational score: 3.9784533977508545 Domain: biomedical Document type: Review Language: en Research is just beginning to elucidate how the various types of glia in the retina adapt to sensing of blurred images in myopia. While neurons have been a traditional focus of ocular research, they are outnumbered by glia, which play vital roles in modulating neuronal processing of visual signals and in regulating eye growth. Our review has identified several areas where future research should deepen and broaden our understanding of how myopia occurs and progresses, thereby identifying potential targets for myopia management. Section title: 8 Future directions Educational score: 4.056968688964844 Domain: biomedical Document type: Study Language: en Studies should attempt to explain the thinning of the inner retina, especially the RNFL and IPL, in myopia. Potential causes include demyelination of ganglion cells and shrinking of their axon diameters, based on results from a chicken model of myopia, in which intraocular ganglion cell axons are myelinated . Whether the same is true in humans or mice, in which ganglion cell axons are not myelinated, remains to be seen. Axon damage may contribute to the blurring of images in myopia by compromising the speed and fidelity of visual signal processing, which requires further investigation. Section title: 8 Future directions Educational score: 4.208946228027344 Domain: biomedical Document type: Study Language: en Future studies, especially those in vivo, should explore whether and how mechanical stretching contributes to myopia progression, and whether Müller cells are involved. The fact that Müller cells penetrate nearly all retinal layers make them well-suited to sensing mechanical stresses . In addition to ocular elongation, myopia involves retinal enlargement, and research should explore the potential contribution of Müller cells here as well. The retina is likely to enlarge though a process more complicated than simple stretching, because retinal thinning occurs primarily in the inner retina, not across all retinal layers. Secretion of MMPs and TIMPs by Müller cells and the reprogramming of these cells may remodel the extracellular matrix of the retina to facilitate enlargement . Section title: 8 Future directions Educational score: 4.133216381072998 Domain: biomedical Document type: Study Language: en Like the retina itself, the vitreous also enlarges in myopia, and this has traditionally been regarded as an automatic “byproduct” of ocular elongation and therefore neglected in the literature. However, studies suggest that osmotic changes due to accumulation of extracellular K + in the myopic eye may lead to transretinal fluid movement that is mediated by aquaporin 4 on Müller cells and that leads to vitreous enlargement. This potential mechanism should be explored in future work, which may also help to explain why the choroid thins in myopia. Section title: 8 Future directions Educational score: 3.936898946762085 Domain: biomedical Document type: Study Language: en Studies are urgently needed into the potential role of retinal microglia in myopia, a topic that has been sorely neglected in the literature. How microglia respond to abnormal visual experiences and the retinal microenvironment in myopia remains unknown. An obvious line of investigation to explore is the involvement of a pro-inflammatory environment, which is known to activate microglia and thereby alter their morphology and behavior . This and other lines of investigation need to examine whether and how microglia contribute to myopia onset and progression. Section title: 8 Future directions Educational score: 3.799981117248535 Domain: biomedical Document type: Review Language: en Ultimately, a major goal in elucidating the roles of retinal glia in myopia is to identify therapeutic targets. To our known, there is not clinic trails or applications targeting retinal glia cells. The standard strategy for controlling excessive axial elongation is to reinforce the posterior sclera with various materials . Another possibility is to reinforce the inner retina or ILM in order to inhibit Müller gliosis . This as well as other mechanical and pharmacological approaches to modulating retinal glia should be explored for controlling myopia progression. | Review | biomedical | en | 0.999998 |
PMC11696180 | Section title: Introduction Educational score: 4.185389518737793 Domain: clinical Document type: Clinical case Language: en Disseminated herpes simplex virus (HSV) infection is a rare but potentially life-threatening condition, especially in immunocompromised individuals, and is associated with high mortality . HSV remains dormant in neural ganglion cells and is reactivated during immunosuppression. Typically, it causes upper respiratory tract infection but occasionally can lead to severe illness needing intensive care unit (ICU) admission. HSV may be isolated in respiratory specimens of many critically ill patients especially those who have been on mechanical ventilation greater than five days. It is difficult to differentiate whether the detection of HSV represents asymptomatic shedding or a true infection responsible for pneumonia . HSV pneumonitis should be suspected in patients with acute respiratory distress syndrome (ARDS) with negative bronchoalveolar bacterial cultures and a negative respiratory viral panel. Here, we present a case of HSV pneumonitis presenting as ARDS and septic shock with a history of transient neutropenia secondary to chemotherapy. Bronchoalveolar lavage (BAL) and blood were positive for HSV PCR. Acyclovir was started with a good clinical response. Section title: Case presentation Educational score: 3.4733636379241943 Domain: clinical Document type: Clinical case Language: en An elderly female patient, aged 72, was admitted to ICU for management of acute hypoxic respiratory failure. Her medical history included hypertension, gastro-esophageal reflux disease, chronic kidney disease stage IIIa (eGFR of 50 mL/min and baseline creatinine of 1.5 mg/dL), and a former tobacco use disorder (cigarette smoking, 30 pack years, quit eight years ago). Additionally, she had recently (three months ago) been diagnosed with stage III rectal adenocarcinoma and was undergoing chemotherapy with the FOLFOX (Folinic acid, Fluorouracil, Oxaliplatin) regimen. Section title: Case presentation Educational score: 3.842806577682495 Domain: clinical Document type: Clinical case Language: en Four days after completing the sixth cycle of chemotherapy, the patient presented to a suburban facility with symptoms of fever, malaise, loss of appetite, abdominal pain, and diarrhea and was subsequently admitted for neutropenic sepsis. Broad-spectrum antibiotics were immediately initiated; however, blood and urine cultures yielded negative results. Due to the progressive worsening of her respiratory status, she required intubation and was subsequently transferred to our facility for further care. Upon arrival, the patient's vital signs revealed a temperature of 101°F, a pulse of 110, a blood pressure of 80/60, and a saturation of 91% with 100% FiO 2 . The physical examination was notable for diffuse crepitations in bilateral lung fields. The initial chest radiographs were consistent with bilateral diffuse consolidation consistent with ARDS. Immediate resuscitation measures were taken, including the placement of a central line and an arterial line. Intravenous fluid resuscitation was done with normal saline at 20 mL/kg. Vasopressors including nor-epinephrine and vasopressin at 0.03 units/hr were initiated. Nor-epinephrine infusion was titrated to a mean arterial pressure (MAP) goal greater than 65 mmHG, requiring 0.3 to 0.4 mcg/kg/min. Initial ABG revealed a pH of 7.19 and PaCO 2 of 38. The bedside echocardiogram indicated a grossly normal ejection fraction of 60-65% and an absence of preload responsiveness by the absence of a significant increase of velocity-time integral with passive leg raise. See Table 1 for laboratory workup. Section title: Case presentation Educational score: 3.9413583278656006 Domain: clinical Document type: Clinical case Language: en The patient was found to be profoundly acidotic and exhibited acute kidney injury, leading to the immediate initiation of continuous renal replacement therapy (CRRT). Mechanical ventilation was managed using low tidal volume ventilation at 6 mL/kg predicted body weight (PBW) and positive end-expiratory pressure (PEEP) titration based on driving pressure. With a PEEP of 12 cmH 2 O, the FiO 2 could be reduced to 60% with a PF ratio of 170, thus making prone positioning unnecessary. As the initial respiratory viral panel was negative, a bronchoscopy was done to obtain bronchoalveolar samples. A subsequent bedside bronchoscopy revealed multiple 2-5 mm erosions with erythematous bases diffusely in the tracheobronchial tree indicative of herpetic tracheobronchitis . Section title: Case presentation Educational score: 3.396728277206421 Domain: clinical Document type: Clinical case Language: en A CT chest scan was then performed to determine the extent of consolidation, which revealed diffuse bilateral consolidations and an interstitial pattern . There was evidence of bilateral pleural effusions, consolidation , and traction bronchiectasis . Mild motion artifacts can be appreciated on all images. However, the respiratory viral panel yielded negative results. Section title: Case presentation Educational score: 3.064622163772583 Domain: clinical Document type: Clinical case Language: en Ventilator settings were extremely high, precluding a lung biopsy, which was, therefore, not performed. Empirical acyclovir therapy was initiated at a dose of 7.5 mg/kg (after adjusting for CRRT with eGFR of 22 mL/min) due to a high clinical suspicion of herpes pneumonitis. Section title: Case presentation Educational score: 4.174549102783203 Domain: biomedical Document type: Study Language: en BAL cytology revealed no tumor cells, with a cell differential of 95% neutrophils, 5% lymphocytes, 0% macrophages, and 0% eosinophils. Additionally, a monolayer preparation and cell block preparation revealed scattered benign and degenerative-appearing squamous and bronchial epithelial cells amid an abundance of acute inflammatory cells. Some of these cells exhibited glassy nuclear inclusions, occasional red granular inclusions, and focal multi-nucleation. The affected cells appeared to be squamous epithelial cells, suggesting a viral cytotoxic effect. While these features were suspicious for a possible HSV infection, other viral causes, including cytomegalovirus (CMV), could not be entirely excluded based on cytologic features alone. Immunohistochemical testing for CMV using an analyte-specific reagent antibody (a cocktail of two mouse monoclonal antibodies, DDG9 and CCH2, diluted 1-200) and heat-induced epitope retrieval (bond ER solution 2 for 20 minutes) with a polymer detection system yielded negative results, with an appropriate control (Leica Microsystems Bond Instrument). Further investigations for immunosuppression, including HIV and immunoglobulin deficiency, were negative, except for a brief episode of neutropenia on the day of presentation, which responded well to filgrastim administered at the outside facility. HIV testing was negative. Subsequently, HSV-1 PCR testing returned positive in both BAL and blood samples, with results available on day 2 of ICU admission. A CT scan of the head and fundoscopy revealed no lesions suggestive of herpes infection. Section title: Case presentation Educational score: 3.5493602752685547 Domain: clinical Document type: Clinical case Language: en Infectious diseases were consulted, and it was recommended to continue the same antiviral therapy for disseminated herpes infection. The patient remained on vasopressors (norepinephrine and vasopressin) for four days to target a MAP of 70 mm of Hg. The course was further complicated by new-onset atrial fibrillation on day 4 of admission, necessitating amiodarone infusion for three days. Beta-blockers and calcium channel blockers were not administered due to the patient's septic shock. Anticoagulation was deemed inappropriate due to diffuse oozing in the tracheobronchial tree. CRRT was successfully terminated on day 4, with the patient experiencing good renal recovery as defined by urine output >500 mL/day. Mechanical ventilation was continued for a total of seven days, after which the patient was successfully extubated and transitioned to a high-flow nasal cannula (30 L/min of flow and 40% FiO 2 ) following a two-hour spontaneous breathing trial. Acyclovir therapy was administered for a total of 14 days, and subsequent HSV PCR testing in the blood sample yielded negative results. The patient was also found to have critical illness neuromyopathy. Ultimately, the patient was discharged to a short-term rehab facility on the 12th day after admission. At the time of discharge, she required 2 L/min of supplemental oxygen. Further discussion with the patient revealed a history of episodes of herpetic labialis (cold sores) in the past, with the most recent occurrence being one week prior to admission. The patient was followed up in the post-ICU clinic for one month and is clinically doing well. Section title: Discussion Educational score: 4.181220531463623 Domain: clinical Document type: Clinical case Language: en This case highlights the challenges in diagnosing and managing disseminated (blood-stream infection) HSV infection in immunocompromised patients. The initial presentation with neutropenic sepsis and acute respiratory failure, coupled with negative blood and urine cultures, posed a diagnostic dilemma. However, bronchoscopy and subsequent testing confirmed herpetic tracheobronchitis and pneumonitis. The prompt initiation of antiviral therapy, along with supportive care, resulted in a favorable outcome. HSV infection most commonly involves the upper respiratory tract, causing herpes labialis (cold sores) or gingivostomatitis and pharyngitis. This is usually due to reactivation during periods of stress, trauma, and fever. Until then, it remains dormant in neural ganglion cells. However, in immunosuppressed individuals (especially cell-mediated immunity), reactivation may result in life-threatening infections . For this reason, most transplant recipients frequently receive prophylaxis with acyclovir. Our patient received chemotherapy for cancer-causing transient neutropenia, which must have caused the reactivation of the dormant herpes virus. The pathogenesis of HSV pneumonia is thought to occur from aspiration of salivary secretions and, during the transit, can result in pharyngo-tracheobronchitis . On further questioning, our patient had a history of herpes labialis occurring one week prior to the hospital admission. Bronchoscopy performed during the ICU admission clearly demonstrated multiple 2-5 mm erosions suggestive of herpetic tracheobronchitis. Herpes simplex pneumonia presents with prodromal symptoms such as fever, myalgias, and GI symptoms. Our patient presented initially with complaints of abdominal pain and diarrhea and, with evidence of neutropenia, was empirically initially treated for neutropenic enterocolitis and received broad-spectrum antibiotics. This was followed by progressive respiratory failure, likely representing herpes pneumonitis. Blood cultures and CT abdomen pelvis were negative. Disseminated herpetic infection should be strongly in patients with neutropenia who do not respond to broad-spectrum antibiotics. In our case, HSV pneumonitis resulted in severe ARDS needing mechanical ventilation. Section title: Discussion Educational score: 4.373404026031494 Domain: biomedical Document type: Study Language: en HSV is also frequently isolated in respiratory specimens of critically ill patients (5-64%), especially with prolonged intubation of >5 days . In many circumstances, this represents a carrier state (referred to as innocent bystander due to asymptomatic shedding) rather than pneumonia. If HSV is the cause of pneumonia rather than being a carrier state, the prognosis is guarded . Unwarranted therapy carries the risk of organ failure due to the drug's narrow therapeutic index, more so in critically ill patients who are on organ support . Therefore, whether to treat a positive HSV sample or not is challenging, especially in critically ill patients. Of note, the traditional respiratory viral panel does not include HSV, and hence, a negative nasopharyngeal swab should not deter from suspecting herpes pneumonia. Serology is rarely useful as it cannot distinguish past from current infection. A bronchoscope is recommended not only to obtain bronchial washings or BAL, which represent true lower respiratory samples, but also the presence of tracheobronchitis like in our case supports early suspicion and almost always warrants acyclovir therapy . HSV PCR should be obtained on blood samples in all cases of HSV pneumonia, as frequently disseminated HSV infection either as a cause or a consequence can be seen. For the same reason, imaging of the head and fundoscopy can establish the degree of dissemination. This is important as the duration of therapy may be different . Our patient also had a positive HSV PCR on a blood sample representing disseminated herpes infection. Although viral cultures were traditionally considered as gold standard, PCR has become the test of choice due to its 100% sensitivity . Like in our case, cytopathological changes seen on BAL almost always warrant therapy. Lung biopsy, if positive can confirm HSV pneumonia, but a negative result does not exclude. In critically ill patients, lung biopsy can be hazardous due to high ventilator settings . Section title: Discussion Educational score: 3.8072445392608643 Domain: biomedical Document type: Other Language: en Acyclovir therapy should be considered in all cases of positive blood samples or positive respiratory samples along with cytopathological changes on BAL or high viral load or if there is no other cause identified as the cause of respiratory failure . In our case, cytopathological changes were not only seen on the BAL sample, but HSV was positive on the blood sample. Without therapy, the mortality rates can be as high as 30-60% . Section title: Discussion Educational score: 3.8573758602142334 Domain: biomedical Document type: Other Language: en There is no evidence to suggest the recommended dose and duration of acyclovir specifically for HSV pneumonitis. Most intensivists employ a dose recommended for HSV encephalitis of 10 mg/kg IV every eight hours. However, the drug is really cleared due to its low protein binding capacity and high water solubility. Hence, the dose must be adjusted if the patient is on renal replacement therapy. We have employed a dose of 7.5 mg/kg in our patient as she was on CRRT . Steroids should be considered in severe ARDS to prevent fibrosis or to treat complications such as organizing pneumonia . However, steroid administration in mild pneumonia can result in fatal hepatitis and dissemination. We have not considered steroid therapy for the risk of dissemination. Section title: Discussion Educational score: 3.8619415760040283 Domain: biomedical Document type: Study Language: en Older age, smoking history, and prolonged intubation are associated with worse outcomes, even in immune-competent critically ill patients . Rather, some studies have shown that a positive HSV sample is a marker of severity regardless of immunocompetence . Mortality of HSV pneumonia is reported at around 60% . Our case has a positive outcome, likely due to earlier initiation of antiviral therapy due to the presence of herpetic erosions on the tracheobronchial tree. Section title: Discussion Educational score: 3.8646609783172607 Domain: clinical Document type: Clinical case Language: en The case described above had multiple circular erosions with an erythematous base in the tracheobronchial tree on bronchoscopy suggestive of tracheobronchitis. In addition, she was intubated for respiratory failure resulting from ARDS caused by HSV. A history of herpes labialis and herpetic tracheobronchitis a week prior, along with CT chest evidence of viral pneumonitis, is consistent with aspiration of salivary secretions as the cause of respiratory failure. Transient neutropenia would have been the risk factor or dissemination including the isolation of HSV-1 in the blood. Section title: Conclusions Educational score: 3.9987804889678955 Domain: biomedical Document type: Other Language: en In patients with neutropenia, even if transient, HSV pneumonitis should be considered in those presenting with ARDS and septic shock, especially if other diagnostic workups remain negative. It is important to note that a respiratory viral panel does not routinely include HSV testing; therefore, a virus-specific PCR test must be requested. Multiple circular erosions on bronchoscopy are almost always indicative of either HSV or CMV, and early initiation of antiviral therapy can reduce mortality. | Clinical case | clinical | en | 0.999993 |
PMC11696245 | Section title: INTRODUCTION Educational score: 2.6330361366271973 Domain: biomedical Document type: Other Language: en Acute injuries of the medial ulnar collateral ligament (UCL) of the elbow are commonly reported in throwing or overhead athletes like baseball, tennis, volleyball or javelin throwers , but are also seen in simple elbow dislocations and fracture dislocations . Section title: INTRODUCTION Educational score: 4.2089009284973145 Domain: biomedical Document type: Study Language: en Tears of the UCL are commonly treated conservatively with braces, tapes and physiotherapy, with avoidance of valgus stress . Surgical treatment is an option for patients with gross instability and/or chronic instability with functional impairment after conservative treatment . Various rehabilitation protocols have been developed to ensure joint congruity and at the same time restore mobility as quickly as possible . To protect against valgus stress, both conservative and post‐operative treatment protocols include the use of a hinged elbow brace . In contrast to the abundant evidence available on the basics of the injury and its various treatment options, there is only very little evidence regarding the use of hinged braces. Biomechanical studies conducted by Manocha et al. suggested that hinged braces are unlikely to provide additional stability during dynamic range of motion in various positions of the elbow, neither after lateral collateral ligament nor UCL injuries . On the other hand, there is one biomechanical study that reported improved valgus stability after UCL injury when a hinged elbow orthosis was applied . Besides the few mentioned studies, there is no further evidence about the absence or presence of a possible protective effect of hinged braces against valgus stress after UCL tears of the elbow. In addition, the protection against passive valgus forces in particular has not yet been sufficiently investigated, which represents an essential factor for successful rehabilitation after UCL injuries. Therefore, the aim of the present study was to investigate the effectiveness of a commercially available elbow orthosis in reducing passive valgus forces after UCL injury and thus to substantiate or invalidate its relevance in the treatment of this pathology. Consequently, the hypothesis of the study is that a hinged elbow orthosis in a 90° fixed flexion position significantly reduces passive valgus forces in the elbow following UCL injuries, while the null hypothesis posits that the orthosis does not have a significant effect on reducing these forces. Section title: Ethical considerations Educational score: 1.2083321809768677 Domain: biomedical Document type: Other Language: en This in‐vitro study was approved by the local institutional review board (Ethical Committee of the Medical Faculty of the University of Cologne—VT ). This study followed the guidelines for experimental investigation with human subjects required by our institution. Section title: Specimen preparation Educational score: 4.056777000427246 Domain: biomedical Document type: Study Language: en For this biomechanical study, eight fresh frozen cadaveric elbow specimens from three male and five female body donors were available. This sample size was based on previous studies with similar designs . Three of the specimens were right arms and five of them were left arms. The mean age at the time of death was 82 years (min. 65 years, max. 87 years). The specimens were stored at −20°C and thawed at room temperature 16–18 h before dissection and biomechanical testing. Fluoroscopic and clinical examinations were performed to exclude specimens with osteoarthritis or signs of previous surgery and trauma. The soft tissue of the proximal humerus and the forearm was preserved. Section title: Biomechanical testing set‐up Educational score: 4.196922302246094 Domain: biomedical Document type: Study Language: en The humeral shaft was secured to a custom‐made testing fixture with an external fixator construction and one additional mounting clamp. The hinged testing fixture was positioned at a 90° angle and was mounted onto a servo‐hydraulic universal testing machine (ZwickRoell). The tested forearms of the specimens were secured in neutral position by two Kirschner wires (K‐wires), which were placed through the radius and the ulna. A mounting bolt was securely fixed to the lateral side of the ulnar shaft 10 cm distal to the centre of rotation. A synthetic wire connected the bolt to the mobile traverse of the testing machine. Reels were used for deflection of the wire. Thereby, upward movement of the mobile traverse resulted in a valgus force. By placing the forearm on a mobile platform, valgus forces could be converted into corresponding horizontal movements . A variant of this biomechanical testing set‐up was used in previous studies . To detect the differences in valgus instability we tracked the horizontal movement of the forearm by using the Optotrak Certus® motion capture system (Northern Digital Inc.). It is a camera‐based tracker, which captures the coordinates of position markers consisting of infrared light‐emitting diodes. The three sensors in a camera are placed slightly apart, so an active marker will be seen by the sensors at slightly different angles. For each marker, these two‐dimensional (2D) sensor data are used to calculate the 3D position coordinates. For data acquisition from the Optotrak system, we used Northern Digital's own ‘First Principles’ software. The corresponding sensor was attached to the K‐wire, which was drilled through the ulnar and radial shaft approximately 20 cm distal to the centre of rotation . All examinations were carried out in 90° flexion. Section title: Biomechanical testing set‐up Educational score: 4.089061260223389 Domain: biomedical Document type: Study Language: en The testing protocol provided for an initial load of 1 N tensile force from the hydraulic machine. Positioning the traction pin on the lateral ulna approximately 10 cm distal to the centre of rotation of the elbow resulted in a traction force of 0.1 N m on the UCL. As a result, the elbow was always brought into the same starting position. The tensile force was then gradually increased to 10, 20 and finally 30 N, resulting in a valgus force of 1, 2 and 3 N m. The horizontal movement of the forearm was detected with the Optotrak system at all four levels, that is, the starting position, at 1, 2 and 3 N m. To test the effectiveness of the hinged brace in reducing valgus forces, three different scenarios (A–C) were prepared. The described testing protocol was repeated three times for each scenario and for each torque three times. Section title: Scenario A Educational score: 2.8393657207489014 Domain: biomedical Document type: Study Language: en In Scenario A, the skin, subcutaneous tissue, ligaments and fascia of the forearm remained intact . The cadaveric elbows were mounted on the testing set‐up as described above, and valgus instability was tested. Section title: Scenario B Educational score: 4.105679035186768 Domain: biomedical Document type: Study Language: en After testing the specimens in the native state, a medial approach to the elbow was performed . The flexors were split but preserved, the posterior and anterior bundles of the UCL were detached from their humeral origin together with the medial capsule, mimicking a ruptured state of the UCL, resulting in valgus instability of the elbow. Valgus instability was confirmed through a clinical examination of the specimen elbow. This involved applying a valgus force to the elbow joint while observing the medial joint opening. The clinical examination was performed by a trained orthopaedic resident who assessed the medial joint line for increased laxity and abnormal movement, indicating instability. This was followed by testing for valgus instability as described above. Section title: Scenario C Educational score: 4.057564735412598 Domain: biomedical Document type: Study Language: en After testing the specimens in the transected state, we applied a hinged elbow brace in a 90° fixed flexion position (medi Epico ROM®s, medi GmbH & Co. KG). The orthosis was secured to the arm using the adjustable strap system and a handpiece designed for a snug fit. This allowed easy adjustment and a custom fit for every specimen tested. The orthosis was positioned in such a way that the centre of rotation of the orthosis was projected onto the idealised centre of rotation of the elbow. According to Graham, the trochlea centre and the centerline of rotation are essentially in line with each other with the elbow flexed 90° . Therefore, the centre of the trochlea was visualised in 90° flexion under fluoroscopy and the elbow orthosis was fitted in such a way that the hinge of the orthosis was in line with the trochlea. Then, the elbow was tested again for valgus instability as described below. Section title: Vector analysis Educational score: 3.8617703914642334 Domain: biomedical Document type: Study Language: en After gathering the three‐dimensional (3D) position coordinates from the Optotrak system for each step of the test protocol, we had each X , Y and Z position of the distal pin and thus we were able to calculate the horizontal movement, that is, the extend of the valgus instability, as mathematical vectors. υ → = x y x . ∣ υ → ∣ = x 2 + y 2 + z 2 . Section title: Vector analysis Educational score: 3.967928886413574 Domain: biomedical Document type: Study Language: en We tested every specimen for every scenario and every level of valgus force three times (i.e., three test repetitions) yielding three different vectors. The mean values of these three vectors were calculated and used in all statistical analyses. The calculated amounts of the vectors were given in millimetres. The vector length in millimetres refers to the horizontal movement of the position marker and represents the corresponding (smaller) movement that occurs due to the medial instability at the medial side of the joint. Section title: Vector analysis Educational score: 3.022784471511841 Domain: biomedical Document type: Study Language: en The relationships between the scenarios and the corresponding summed vectors were then calculated as factors, that is, B/A and C/B as relative deviations, corresponding to a multiplicative model. Section title: Statistical analysis Educational score: 4.028505325317383 Domain: biomedical Document type: Study Language: en The data collected were analysed using the Statistical Package for the Social Sciences statistical programme. Normal distribution was tested by Kolmogorov–Smirnov. We report the median (interquartile range). To determine differences between the groups we performed a two‐way repeated measures analysis of variance. The effect size was calculated according to Cohen. A p ≤ 0.05 was considered to be statistically significant. Intra‐specimen variability was measured using the intraclass correlation (ICC) according to Shrout and Fleiss . ICC benchmarks were used as proposed by Cicchetti (poor: ICC < 0.4; moderate: 0.4–0.59; good: 0.6–0.74 and excellent: 0.75–1.0) . Section title: RESULTS Educational score: 3.291316509246826 Domain: biomedical Document type: Study Language: en The pooled mean value for the magnitude of the vector length is shown in Table 1 . The smallest vector length was in Scenario A at 1 N m. The highest vector length was in Scenario B at 3 N m. There was a significant difference between the vector lengths within Scenarios B and C. The post hoc test showed that in Scenarios B and C, only the difference between 1 and 3 N m was significant ( p = 0.041 and p = 0.014). For an overview of these results, see Table 1 and Figure 4 . Section title: RESULTS Educational score: 2.1522905826568604 Domain: biomedical Document type: Study Language: en The mean values of the vector lengths for every single specimen, scenario and torque are shown in Table S1 . Section title: RESULTS Educational score: 3.852254867553711 Domain: biomedical Document type: Study Language: en The ratio of the vector length of Scenario A in comparison to Scenario B was on average 0.63 (95% CI: 0.47–0.78). The ratio between Scenarios B and C was on average 2.77 (95% CI: 1.42–4.13). Comparing Scenario A with Scenario C, the ratio was on average 2.07 (95% CI: 0.63–3.51). Section title: RESULTS Educational score: 2.469370126724243 Domain: biomedical Document type: Study Language: en The vector length ratios were statistically significantly different depending on the applied force ( p = 0.038), but not for the respective scenario (n.s.). There was no significant influence of the interaction between the scenario and the force (n.s.). Section title: RESULTS Educational score: 2.634951114654541 Domain: biomedical Document type: Study Language: en The ICC for the intra‐specimen variability is depicted in Table 2 . Section title: DISCUSSION Educational score: 4.140048503875732 Domain: biomedical Document type: Study Language: en The main finding of this study is that the hypothesis—that a hinged elbow orthosis significantly reduces passive valgus forces in the elbow following UCL injuries—is not supported by the data and, therefore, has to be rejected. Although there was a tendency for the brace to mitigate valgus forces, this effect did not reach statistical significance. The results show that the dissection of the UCL created an ulnar instability with increased horizontal deflection by 37%. The results further show a tendency, that when applying a hinged elbow brace to the elbow with valgus instability, the horizontal deflection was reduced. While the results presented in Table 2 suggest a large inter‐specimen variability, the calculation of the ICC for the intra‐specimen variability showed values of excellent quality in seven of the eight specimens. This shows the validity of the experiment carried out to record the effect of valgus instability and its change. Therefore, it is possible that the inter‐specimen variability is due to the different biological nature of the specimens available for testing. Section title: DISCUSSION Educational score: 4.199230670928955 Domain: biomedical Document type: Study Language: en The evidence about the effectiveness of elbow orthoses to reduce valgus instability remains controversial. One biomechanical study reported by Pincivero et al., involved designing a custom orthosis for a javelin thrower with unilateral MCL insufficiency. The authors applied varying degrees of valgus force to the athlete's elbow. They found that the hinged elbow orthosis restored valgus stability on both the injured and uninjured sides, although its effectiveness decreased as the valgus force increased . In contrast, there is a biomechanical cadaver study in which the authors used a custom‐built simulator to study seven cadaver elbows in active and passive motion in the physiological state, with a torn UCL and with an elbow brace attached to the elbow with a torn UCL . The authors found that the hinged elbow brace had no beneficial effects on valgus instability during passive movement and even increased valgus instability during active movement. In that experiment, the elbow fulfilled the whole range of motion from full extension to full flexion, active and passive. The authors addressed the issue that as the elbow flexes from a fully extended position, the humerus undergoes internal rotation. As the elbow approaches full flexion, the humerus externally rotates, causing the elbow to shift from a more valgus to a more varus position during flexion . Under these circumstances, the hinged elbow brace did not support valgus stability. Our data, on the other hand, demonstrate contradicting findings. The scenarios in our study were designed with a fixed 90° flexion position of the elbow. Valgus force of 1–3 N m was applied so that this reflects the approximate forces that can be assumed in light everyday movements, such as sliding an object or drinking . The presented data show that in these specific situations, with rather a static movement without full flexion or full extension and with just a little force applied to the elbow, the hinged elbow brace may offer additive support of the medial elbow. Although the amount of force reduction does not reach the level of the native UCL, there is an indication of reduction with a close approximation to the native state. However, it is important to note that the brace was fitted perfectly every time under the study conditions. Therefore, the results can only show the protective effect under these circumstances. It is known from clinical practice that braces can slip out of place or be unintentionally put on incorrectly by patients. This may lead to a malalignment of the mechanical axis of the orthosis and the anatomic flexion–extension axis of the elbow, which may exert harmful leverage forces on the elbow joint . This was also discussed as a possible reason for an increased valgus instability with an applied hinged elbow orthosis during active movement after UCL rupture in the previously mentioned cadaveric study by Manocha et al. . The problem of harmonising the mechanical axis of the orthosis and the anatomical axis of the elbow could be further complicated by the fact that the flexion–extension axis of the elbow is altered by a UCL injury . However, the results of our study show that if the mechanical axis of the orthosis and the anatomical axis of the elbow are brought into alignment, the orthosis could have possibly a protective effect against valgus forces. The risks of malalignment might be avoided by using a handrest as well as an adequate introduction to brace application by the orthopaedic technician. It has also been shown that after surgical reconstruction using modern surgical techniques of the UCL, the anatomical flexion–extension axis is restored to approximately the physiological state and is no longer subject to variability as described above . Therefore, complications due to an altered axis of the elbow following a UCL rupture should be mitigated after surgical treatment and the alignment of the mechanical axis of the orthosis with the elbow axis should be facilitated. This could indicate an increased efficacy or relevance of orthotic therapy after surgical treatment compared to the use of a hinged elbow brace in conservative therapy after a UCL injury. Beyond that, an improved orthosis design that ensures the correct alignment of the axes could improve the treatment with orthoses, which has also been suggested by other authors . Section title: DISCUSSION Educational score: 4.178053855895996 Domain: biomedical Document type: Study Language: en In addition to the lack of support of the orthoses for the elbow with valgus instability during movement of the joint, the previous biomechanical study by Manocha et al. showed a reduction of valgus instability due to active movement of the muscles . This stresses the necessity of correct execution of exercises and movement patterns in general after UCL injuries. Systematic reviews and meta‐analyses in the field of knee surgery have shown that neuro‐muscular status plays a key role in the primary and secondary prevention of ligamentous knee injuries . Various training programmes to improve neuro‐muscular functionality have shown their effectiveness in preventing these injuries. There are no equivalent examinations for ligamentous injuries of the elbow. However, muscle training is a key element of rehabilitation programmes in elbow surgery , because the flexor and extensor muscles of the forearm are secondary stabilisers of the elbow joint . Hence, similar effects, like in the prevention of knee injuries, can be surmised. Therefore, we do not see our study results as contradicting the previous findings on the effects of a hinged elbow brace during elbow joint movement, but rather see our study as a completely different area of interest. While the effects mentioned above are important during rehabilitation programmes, protection from passive valgus forces is key for supporting healing during daily life. Most post‐operative protocols as well as the non‐operative protocols suggest non‐weight bearing or pausing the causative activities and different kinds of restriction of movement for at least six weeks . While patients can protect their injured elbow in this period of time from valgus forces through conscious movement sequences, it can still happen repeatedly in everyday life that some movements of daily life are carried out less consciously or completely unconsciously. This may result in passive valgus forces affecting the elbow. The data presented in this study suggests that an elbow orthosis, at least in a 90° fixed flexion position, may reduce these described passive forces to the elbow in these situations of daily life, when the orthosis is in correct position and the mechanical and anatomical axis are correctly aligned. Section title: LIMITATIONS Educational score: 4.154806613922119 Domain: biomedical Document type: Study Language: en The present study has several limitations. First, the small sample size may harbour the risk of a type two error. This is a well‐known and common drawback of biomechanical research. The sample size of eight cadaveric elbows was chosen to balance the feasibility of obtaining and preparing fresh frozen specimens with the need for sufficient data to test differences in valgus instability across the test scenarios. This number aligns with previous biomechanical studies that have demonstrated reliable and reproducible results using similar sample sizes . The chosen sample size also considers the high cost and limited availability of cadaveric specimens, making it a practical and justifiable choice for this study. Second, this study as well as most biomechanical studies face the problem of a big age difference of the older cadaver samples compared to the usually much younger patient population of interest. In addition, a disadvantage of this study is that the experiment was carried out in 90° flexion only. Other angular dimensions were not examined. The contradictory high vector length observed in the last specimen for scenario A could be due to pre‐test instability, tracker mobilisation, or specimen fixation failure, all of which could have led to erroneous measurements. Another limitation is the measurement error of the Optotrak system used. The accuracy provided is 0.1 mm although there could be a difference to the actual vectors and the vectors measured . Finally, biomechanical study conditions do not accurately reflect the physiological reality that is to be investigated in the studies. Section title: CONCLUSION Educational score: 4.106687068939209 Domain: biomedical Document type: Study Language: en The hypothesis—that a hinged elbow orthosis significantly reduces passive valgus forces in the elbow following UCL injuries—is not supported by the data and therefore has to be rejected. Nevertheless, the study demonstrates a tendency that a hinged elbow brace may reduce passive valgus forces following UCL rupture, thereby possibly offering a potential protective effect against valgus instability in specific static conditions. While there is variability among specimens, the consistent intra‐specimen results validate the experimental set‐up. The study highlights the importance of proper brace alignment with the elbow's anatomical axis to maximise efficacy. However, the protective effect is context‐dependent and may not extend to dynamic movements, emphasising the need for patient education and possibly improved orthosis design. Section title: AUTHOR CONTRIBUTIONS Educational score: 0.9752180576324463 Domain: other Document type: Other Language: en Kai Hoffeld : Data curation; formal analysis; investigation; writing—original draft. Christopher Wahlers : Data curation; formal analysis; investigation; software. Jan P. Hockmann : Data curation; formal analysis; software; visualisation; writing—review and editing. Sebastian Wegmann : Project administration; resources. Nadine Ott : Funding acquisition; methodology; resources. Kilian Wegmann : Conceptualisation; funding acquisition; resources; writing—review and editing. Lars Peter Müller : Supervision; validation; writing—review and editing. Michael Hackl : Conceptualisation; methodology; supervision; writing—review and editing. Section title: CONFLICT OF INTEREST STATEMENT Educational score: 0.8662789463996887 Domain: other Document type: Other Language: en The authors declare no conflicts of interest. Section title: ETHICS STATEMENT Educational score: 1.0763541460037231 Domain: biomedical Document type: Other Language: en Ethical approval for this study was given by the Institutional Review Board of the University Cologne (VT ). Due to cadaveric character of this study, no informed consent was needed. | Other | biomedical | en | 0.999996 |
PMC11696252 | Section title: INTRODUCTION Educational score: 4.080806732177734 Domain: biomedical Document type: Study Language: en A major risk factor for knee osteoarthritis (OA) development and progression is when the load on the joint surfaces exceeds the mechanical properties of the cartilage . A high joint load can be caused by a high body mass index (BMI) , redistribution of the joint loading after a meniscal tear and/or meniscectomy or as a result of altered loading patterns in the case of an anterior cruciate ligament (ACL) injury . As such, ACL injury is a well‐known risk factor for both tibiofemoral (TF) and patellofemoral (PF) OA in younger individuals, that is, posttraumatic OA . The risk for posttraumatic OA increases when the meniscus is injured in addition to the ACL, and especially after meniscectomy . Section title: INTRODUCTION Educational score: 4.341612815856934 Domain: biomedical Document type: Study Language: en In studies of nontraumatic knee OA, anatomical variations in the hip and in knee alignment have been reported to be associated with compartment‐specific OA . The mechanical alignment of the knee, measured as the hip‐knee‐ankle angle (HKA), affects load transmission in the TF joint and alters patella kinematics and contact forces . There is ample evidence that varus and valgus malalignment of the knee is associated with knee OA progression of the medial and lateral TF compartment, respectively . Varus malalignment has furthermore been associated with the development of medial TF OA , whereas no such association was reported for valgus alignment and lateral TF OA development. No association has been found between knee alignment, changes in knee cartilage volume and TF OA development . It has been suggested that differences in hip and pelvic geometry can explain sex‐related differences in OA, with an increased incidence of lateral TF OA compared to medial TF OA in females compared to males . Convincing evidence that posttraumatic OA development after ACL injury is associated with mechanical alignment of the injured leg has not been documented , and the relationship between hip anatomy and knee OA development after ACL injury is unknown. Section title: INTRODUCTION Educational score: 4.129951477050781 Domain: biomedical Document type: Study Language: en The aim of the present study was to investigate if hip and knee alignment assessed 2 years after ACL injury was associated with compartment‐specific OA of the injured knee 3 years later . We hypothesized that a higher neck‐shaft angle (NSA) and valgus alignment of the ACL injured knee would be associated with the development of lateral TF OA, whereas a smaller NSA and varus alignment would be associated with medial TF OA. In a secondary analysis, we aimed to assess the association between alignment at the 2‐year follow‐up and partial meniscectomy performed up to the 2‐year follow‐up. Section title: METHODS Educational score: 3.9232242107391357 Domain: biomedical Document type: Other Language: en Active young adults (18–35 years, 26% women) with an ACL injury within four weeks before examination were recruited for the knee ACL, nonsurgical versus surgical treatment (KANON) trial . Exclusion criteria were previous ACL injury to the knee, total collateral ligament ruptures, extensive meniscal fixation, professional athletes with a Tegner score of 10 and inactive people with a Tegner score <5. All patients were recruited from Skåne, the southern region of Sweden. A total of 121 patients were included and 62 were randomized to undergo rehabilitation with early ACL reconstruction and 59 to undergo rehabilitation and optional delayed ACL reconstruction . Of the 59 to undergo rehabilitation and optional delayed ACL reconstruction a total of 23 underwent ACL reconstruction, on average 347 ± 124 days from baseline . Section title: METHODS Educational score: 4.1630473136901855 Domain: biomedical Document type: Study Language: en At the 2‐year follow‐up, full‐limb radiographs were obtained of the index leg of all the participants, with the radiographed leg in weight‐bearing and in slight knee flexion. The radiographs were obtained using a General Electric Prestige II (General Electric Medical Systems). The HKA and the NSA were measured using the RadiAnt digital imaging and communications in medicine Viewer with an accuracy of 0.1° . The HKA was assessed by drawing a line from the centre of the femoral head to the mid‐distance of the tibial spines. An additional line was drawn from the centre of the trochlea of talus to the centre of the tibial spines. The HKA angle was defined as the medial angle at the intersection of the two lines . The NSA was defined as the angle between the femoral neck axis and the femoral long axis, with the femoral neck axis defined as the line between the centre of the femoral head and the femoral neck centre. The femoral long axis was assessed by drawing a line through two points; one in the centre of the femoral condyles and the other in the middle of the femoral shaft, just below the lesser trochanter . Section title: METHODS Educational score: 4.149420738220215 Domain: biomedical Document type: Study Language: en At the 5‐year follow‐up, radiographs of the injured knee were performed. Axial view of the PF joint was acquired as previously described, according to Ahlbäck . TF radiographs were acquired using a modified version of the Lyon‐Schuss projection, where the subject stands with equal weight bearing on each leg . Radiographs were graded according to the OA research society international atlas and radiographic OA was defined based on any of the following three criteria in either of the two TF compartments or in the PF compartment that was present: (a) joint space narrowing (JSN) grade ≥2; (b) the sum of the osteophyte compartment grades ≥2 or (c) JSN grade of 1 together with a grade 1 osteophyte within the same compartment. These criteria approximate radiographic knee OA grade ≥2 according to the Kellgren and Lawrence scale . Section title: METHODS Educational score: 4.18336296081543 Domain: biomedical Document type: Study Language: en At the 5‐year follow‐up, 116 of the 121 individuals included in the KANON trial underwent full‐limb radiography of the injured leg. Because of bad film quality, no measurements were made on one of the radiographs, leaving measurements of 115 ACL‐injured legs. The 115 patients with full‐limb radiographs of the injured leg were included in the present analysis . To test intrarater variability of HKA and NSA assessments, 20 radiographs were re‐measured (1 month between measurements) and the intraclass correlation was 0.98 (95% confidence interval [CI] 0.97–0.99) for the HKA and 0.95 (95% CI 0.86–0.98) for the NSA. Section title: METHODS Educational score: 2.65368914604187 Domain: biomedical Document type: Study Language: en All 115 subjects completed the knee injury and OA outcome score for patient‐assessed outcomes at the 5‐year follow‐up to assess how the patient perceived pain, other symptoms, function in sports and recreation and knee‐related quality of life, during the previous week . Section title: Statistics Educational score: 4.033334732055664 Domain: biomedical Document type: Study Language: en Statistical package for the social sciences (SPSS) statistical software (version 22, SPSS Inc.) was used for all analyses. Continuous variables were reported as mean (SD) and differences between groups were tested with the Student's t test. All continuous variables were normally distributed. Differences between groups in dichotomous variables were tested with the χ 2 test. Analysis of covariance was used to test those variables which were significantly associated with OA in the crude analysis. Adjustments were made for sex, age, BMI, randomization, partial medial meniscectomy and partial lateral meniscectomy of the injured knee. In a secondary analysis, the knees were categorized as varus (HKA‐angle ≤ 178°), neutral (HKA‐angle 179–181°) and valgus (HKA‐angle ≥ 182°) and logistic regression was used to test how varus and valgus alignment in relation to neutral alignment 2 years after ACL injury related to the prevalence of OA 5 years after ACL injury. Section title: RESULTS Educational score: 4.027005672454834 Domain: biomedical Document type: Study Language: en A total of 115 patients were included in the study (Table 1 ). The patients had a mean BMI of 24.1 and a mean age of 26.2 years (26% were female); 20% of the patients underwent partial medial meniscectomy, 30% underwent partial lateral meniscectomy and 17% underwent meniscal fixation before the 2‐year follow‐up. Neither the HKA nor the NSA was statistically associated with randomization to undergo rehabilitation with early ACL reconstruction versus undergoes rehabilitation and optional delayed ACL reconstruction (Supporting Information S1: Table S1 ). Section title: Association between NSA and HKA angle 2 years after ACL injury with OA 5 years after ACL injury Educational score: 4.1249566078186035 Domain: biomedical Document type: Study Language: en In patients who had developed medial TF OA at the 5‐year follow‐up after ACL injury, the HKA angle of the injured leg at the 2‐year follow‐up was 176.4° compared to 178.7° in patients who had not developed medial TF OA; mean difference, −2.3° (95% CI −4.2° to −0.4°) (Table 2 ). In patients who had developed medial TF OA, the NSA of the injured leg at the 2‐year follow‐up was 124.7° compared to 129.3° in patients who had not developed medial TF OA; the mean difference was −4.6° (95% CI −7.9° to −1.1°). In the adjusted analysis model, the HKA and NSA angles remained statistically significantly different between patients who had developed medial TF OA and those that had not developed medial TF OA (Table 2 ). No statistically significant association was observed between the NSA or HKA at the 2‐year follow‐up and lateral TF OA, nor PF OA, at the 5‐year follow‐up (Table 2 ). Section title: The association between alignment and meniscectomies at the 2‐year follow‐up Educational score: 4.155574798583984 Domain: biomedical Document type: Study Language: en Of the 115 patients included in the present study, 23 patients (20%) underwent a partial meniscectomy of the medial meniscus, and 34 patients (30%) underwent a partial meniscectomy of the lateral meniscus in the ACL injured knee between baseline and the 2‐year follow‐up. The HKA was not statistically different ( p = 0.27) between subjects that had undergone a medial partial meniscectomy (mean, 177.2°; standard deviation [SD], 3.1°) and subjects that did not have a medial partial meniscectomy (mean, 177.9°; SD, 2.9°). The HKA was not statistically different ( p = 0.23) between subjects that had undergone a lateral partial meniscectomy (mean, 177.3°; SD, 2.6°) and subjects that did not have a lateral partial meniscectomy (mean, 178.0°; SD.3.1°). Also, the NSA was not statistically different ( p = 0.43) between subjects that had undergone a medial partial meniscectomy (mean, 128.7°, SD.4.7°) and subjects that did not have a medial partial meniscectomy (Mean 129.6°; SD, 5.2°). The NSA was not statistically different ( p = 0.51) between subjects that had undergone a lateral partial meniscectomy (mean, 129.0°; SD, 6.0°) and subjects that did not have a lateral partial meniscectomy (mean, 129.7°; SD, 4.8°). Section title: Varus, neutral and valgus limb alignment and the association with knee OA Educational score: 3.077646255493164 Domain: biomedical Document type: Study Language: en In a secondary analysis, using HKA measurements from the 2‐year follow‐up, patients were characterized into varus, neutral or valgus knee alignment. Section title: Varus, neutral and valgus limb alignment and the association with knee OA Educational score: 4.072294235229492 Domain: biomedical Document type: Study Language: en Of 64 patients with varus knees, 15 had TF OA (nine medial and six lateral) and 12 had PF OA of the injured knee at the 5‐year follow‐up; none of the 30 patients with neutral alignment had OA in the TF joint and 7 had PF OA; one of the 21 patients with valgus knees had lateral TF OA of the injured knee and three had PF OA (Table 3 ). Compared to neutral alignment, there was no statistically significant association between varus or valgus alignment at the 2‐year follow‐up and TF or PF OA at the 5‐year follow‐up (Table 3 ). Section title: The association between alignment and symptoms at 5 years Educational score: 3.805917501449585 Domain: biomedical Document type: Study Language: en Neither NSA nor HKA of the ACL‐injured knee measured at 2 years was statistically significantly associated with KOOS outcomes at the 5‐year follow‐up (Supporting Information S1: Tables S2 and S3 ). Section title: DISCUSSION Educational score: 4.032258987426758 Domain: biomedical Document type: Study Language: en In subjects, who have had an acute ACL rupture 2 years earlier, we found an association between a smaller NSA and HKA (more varus) of the ACL injured leg and development of radiographic medial TF OA 3 years later. We found no statistically significant association between alignment and the risk of lateral TF OA or PF OA. Section title: DISCUSSION Educational score: 3.850221872329712 Domain: biomedical Document type: Review Language: en Injury to the ACL is associated with an increased risk of posttraumatic OA . Posttraumatic OA after ACL injury is multifactorial. Factors such as meniscal injury, damage to the TF cartilage and older age at the time of surgery have been reported to associate with developing posttraumatic OA . Regarding ACL reconstruction and posttraumatic OA, randomized controlled trials have suggested no essential differences in radiographic OA between those with or without surgical reconstruction . Section title: DISCUSSION Educational score: 4.524814128875732 Domain: biomedical Document type: Study Language: en The hip geometry affects the hip abductor lever arm and moment, where a short lever arm (coxa valga) is associated with a reduced abductor moment, less hip abductor strength, increased dynamic knee valgus and increased lateral TF loads during gait. Contrarily, a long lever arm (coxa vara) will increase the adduction moment at the knee and medial TF loads during gait . In line with their hypotheses, Boissonneault et al. reported that knees with lateral OA had a larger NSA, whereas knees with medial OA had smaller NSA. The NSA influences the abductor angle and the abductor lever arm, thereby also affecting the force generated by abductors and adductors at the hip and loads at the knee during gait . A large NSA and thus a shorter abductor lever arm of the hip leads to a decreased mechanical advantage of the abductor muscles . This can lead to the fact that hip adduction is promoted and the dynamic knee alignment changes to valgus, decreasing the knee adduction moment, thereby increasing lateral TF loads and promoting lateral OA of the knee. Also, it was suggested that a shorter lever arm leads to an imbalance in muscular activity during gait. To compensate for the anatomical prerequisites, the abductor muscles and tensor fascia lata passing the knee laterally during loading increase their activity trying to compensate for the shorter lever arm, thus increasing the force applied on the lateral compartment . With a smaller NSA and thus a longer lever arm, it was speculated that hip abduction is promoted, and the dynamic knee alignment changes to varus, increasing the knee adduction moment, thereby increasing medial TF loads and promoting medial OA of the knee . In the literature, strengthening of the hip abductors is shown to decrease pain and improve functional scores in patients with medial TF OA patients . Section title: DISCUSSION Educational score: 4.18079948425293 Domain: biomedical Document type: Study Language: en Varus alignment of the knee has been proposed as a risk factor for posttraumatic OA after ACL injury . Compared to neutral alignment of the knee, varus and valgus alignment shifts the load distribution of the joint where increasing varus alignment leads to increasing loads of the medial compartment and increasing valgus alignment leads to increasing loads of the lateral TF compartment . This, in combination with altered loading of TF and PF cartilage caused by the meniscal damage/meniscectomies and decreased stability in anterior‐posterior and internal‐external rotation can cause increased loads on surfaces previously not exposed to these forces, thereby increasing the risk of OA development . Regarding nontraumatic OA, both knee alignment and hip geometry have been reported to affect the risk of OA development and progression, by affecting the compartment‐specific loads in the TF and PF joints . Section title: DISCUSSION Educational score: 4.102101802825928 Domain: biomedical Document type: Study Language: en In the present study, we found that the HKA angle was associated with the development of radiographic TF OA of the medial compartment, but not with TF OA of the lateral compartment. These findings are somewhat different from what has been observed in nontraumatic OA, where valgus alignment is a risk factor for lateral TF OA development . When categorizing patients as varus, neutral and valgus aligned, we found that all patients with medial TF OA at the 5‐year follow‐up had varus alignment at the 2‐year follow‐up. Interestingly, none of the patients of the present study with neutral alignment had any radiographic signs of TF OA. Nevertheless, there was no statistically significant difference between the groups regarding the risk of compartment‐specific OA (varus vs. neutral and valgus vs. neutral). The low number of patients who had developed medial‐ and lateral TF OA at the 5‐year follow‐up ( n = 9 for medial and n = 6 for lateral) indicate that those results may be attributable to a type II error, suggesting that larger‐sized future studies on this topic are warranted. Due to the exploratory nature of the present study, no prestudy power calculations were performed. Section title: DISCUSSION Educational score: 3.9557602405548096 Domain: biomedical Document type: Study Language: en To sum up, preexisting varus alignment of the hip and knee resulting in higher medial TF loads could alter ACL injury (regardless of reconstruction), associated instability and altered cartilage loading lead to a rapid development of medial TF OA. Section title: DISCUSSION Educational score: 2.7237327098846436 Domain: biomedical Document type: Study Language: en PF loads may also be affected by knee alignment and hip geometry . However, in the present study, we found no such associations. Previous studies have suggested that subjects with a varus knee alignment are twice as likely to develop medial PF OA . Section title: DISCUSSION Educational score: 4.183871269226074 Domain: biomedical Document type: Study Language: en One limitation to our study is the low prevalence of compartment‐specific TF OA and PF OA, suggesting a high risk of statistical type II errors. Also, investigating the HKA angle of the ACL‐injured knee, measured at the two‐year follow‐up is another limitation, since several factors associated with the ACL injury could alter the HKA angle of the injured leg in varus or valgus direction . Factors such as meniscal pathology, bone attrition, osteophytes and ligament damage may in addition to cartilage loss contribute to the knee alignment . Hence, early OA changes which may have developed already 2 years after the ACL injury could affect knee alignment, thereby biasing our findings. As regards the NSA, it decreases from around 160° at birth to an angle of around 130° ahead of skeletal maturity . The radiographic assessment of the NSA can be influenced by the rotation of the hip. Without correction for the rotation of the hip, there is a 1° margin of error in the NSA . The NSA and HKA were measured on full‐limb radiographs using a standardized protocol, hence limiting the risk of major discrepancies in hip rotation between patients. To certify that the radiograph was acquired with the knee in a straight frontal view, fluoroscopic guidance was used in the lateral view to ascertain that the posterior aspect of the femoral condyles was aligned. Previous investigations have shown that, with regard to the femoral transepicondylar and anterior‐posterior axes, the posterior femoral condylar tangent is internally rotated 3‐6° . Hence, the full‐limb radiograph of the hip, in a view perpendicular to the posterior femoral tangent, will be in approximately 3–6° of hip external rotation. Because of the low number of subjects with OA, it was not possible to separately investigate men and women. Section title: DISCUSSION Educational score: 3.2325785160064697 Domain: biomedical Document type: Study Language: en The strengths of our study include the use of standardized full‐limb radiographs to measure the HKA and NSA, the prospective study design and the low loss to follow‐up. Section title: CONCLUSION Educational score: 4.042327880859375 Domain: biomedical Document type: Study Language: en We found that a smaller NSA and HKA of the ACL injured leg 2 years after injury was associated with the development of radiographic medial TF OA 3 years later. These findings indicate that a small NSA and HKA by leading to increased medial TF loads may increase the risk of medial TF OA development after ACL injury, regardless of ACL reconstruction or not. Section title: AUTHOR CONTRIBUTIONS Educational score: 0.891305148601532 Domain: other Document type: Other Language: en Literature review, statistical analysis and manuscript writing/editing : Henrik Nilsson. Literature review, interpretation of data and manuscript writing/editing : Martin Englund. Data collection, interpretation of data and manuscript writing/editing : Richard Frobell. Data collection, literature review and manuscript writing/editing : L. Stefan Lohmander. Interpretation of data and manuscript writing/editing : André Struglics. Study design, literature review, statistical analysis and manuscript writing/editing : Per Swärd. The final manuscript was read and approved by all authors. Section title: CONFLICT OF INTEREST STATEMENT Educational score: 0.8662789463996887 Domain: other Document type: Other Language: en The authors declare no conflicts of interest. Section title: ETHICS STATEMENT Educational score: 2.2689177989959717 Domain: biomedical Document type: Study Language: en Our study is a post hoc analysis of an RCT, the KANON trial . The Lund University ethics committee approved the study, all participants signed informed consent. | Study | biomedical | en | 0.999997 |
PMC11696254 | Section title: INTRODUCTION Educational score: 3.9809482097625732 Domain: biomedical Document type: Study Language: en The normal anterior cruciate ligament (N‐ACL) could be divided into not only two but three fibre bundles: anteromedial bundle medial part (AMM), anteromedial bundle lateral part (AML) and posterolateral bundle (PL) . In the anatomical triple bundle ACL reconstruction , it is desirable to place these three bundle grafts so that they run equivalent to those of N‐ACL. However, there is still no consensus in the tunnel position to achieve this goal, while the tunnel position affects the kinematics of the knee, and its malpositioning leads to poor clinical outcomes, including residual instability and/or graft failure . Section title: INTRODUCTION Educational score: 2.4273688793182373 Domain: biomedical Document type: Other Language: en While the location of the tibial tunnels is almost agreeable, the position of the femoral tunnels has still been controversial . Some advocated that the tunnels should be placed on the posterior part of the attachment area behind the resident's ridge (RR) , while others have been claiming those created on the resident's ridge (OR) . Section title: INTRODUCTION Educational score: 4.100175380706787 Domain: biomedical Document type: Study Language: en In recent years, a technique for matching bone images of computed tomography (CT) and those on magnetic resonance imaging (MRI) has become available, and this has become possible to project a virtual ACL graft (VACLG) on a CT model onto MRI . This study aimed to compare the orientation of N‐ACL on MRI, BR‐VACLG/the virtual ACL graft via the femoral tunnels created behind the RR on the three‐dimensional (3D) CT and OR‐VACLG/the graft via the femoral tunnels created on the RR in the same knee. While some claim the midsubstance insertion/the resident's ridge as the tunnel location for anatomical graft placement based on macroscopic observation , other histological study suggests the area behind the ridge is more appropriate . Thus, our hypothesis was that BR‐VACLG on the 3D CT showed the closer orientation to N‐ACL on MRI. Section title: Subjects Educational score: 2.3614425659179688 Domain: biomedical Document type: Study Language: en The subjects included normal left knees of healthy volunteers without a surgical or fracture history of either knee or suspected ligament instability based on manual examination or MRI. Informed consent was obtained from all the subjects involved, and the study protocol received the approval of our institution's institutional review board for human subject research. Section title: Scans and virtual ACLGs Educational score: 4.176508903503418 Domain: biomedical Document type: Study Language: en As virtual ACL grafts, triple‐bundle grafting was adopted to closely mimic the native ACL. On 3D CT images (Discovery CT 750HD; General Electric) with a slice thickness of 0.625 mm in the knee extension position, BLs such as the resident's ridge (RR), the lateral bifurcate ridge, the apex of the deep cartilage, Parsons’ knob (anterior ridge: AR), the medial intercondylar ridge (MIR) and the central intercondylar ridge (CIR), were identified on both the femur and tibia . In accordance with previous reports, the area posterior to the RR on the 3D CT lateral image was defined as the femoral side attachment of the BR‐VACLG . The proximal and distal parts of the lateral bifurcate ridge were defined as the AM and PL fibre attachment areas, respectively . A 7‐mm‐diameter circle was created in each fibre attachment area, and their centres were defined as F‐AM and F‐PL, respectively . On the tibial side, the attachment area was defined as posterior to the AR , lateral to the MIR, and medial to the CIR . Three regular circles with a diameter of 5 mm were created in the attachments of AMM, AML and PL fibres within the attachment area, and the centres of each circle were designated T‐AMM, T‐AML and T‐PL, respectively . The lines connecting the centre points of each bundle's femoral and tibial attachments were defined as VACLG . For comparison, the other type of VACLG: OR‐VACLG was created, in which F‐AM and F‐PL were placed on the RR, and the tibial side was placed in the same manner as the BR‐VACLG . Section title: Scans and virtual ACLGs Educational score: 4.044773101806641 Domain: biomedical Document type: Study Language: en 3D MRI (EXCELART Vantage 1.5 T; Toshiba) was obtained on the same knee in the same limb position with the following protocol: T2* sequence, 512 × 512‐pixel, slice thickness 1 mm. CT‐MRI image matching system (Aquarius iNtuition viewer; TeraRecon, Inc.) was used to match the CT and MRI bones. The VACLGs were created on the MRI by projecting the centre point of each fibre bundle plotted on the CT onto the MRI. Section title: Scans and virtual ACLGs Educational score: 4.113532066345215 Domain: biomedical Document type: Study Language: en A validation test of CT‐MRI matching was performed with six randomly selected knees. In each knee, the gaps between the bottom point of the trochlear groove and the top of the entrance of the intercondylar notch on the axial slice, between the anterior and posterior borders of the femoral bone on the coronal slice, between the medial and lateral borders of the femoral bone on the sagittal slice were measured on both CT and MRI when matched by three orthopaedic surgeons (H.Y., R.Y. and T.H.) . The mean gap for 36 points (six points each in six knees) was 0.44 ± 0.23 mm, which was comparable to previous reports . Reliability calculations were performed for the bone matching between CT and MRI, and the intra‐observer and inter‐observer intra‐class correlation coefficients (ICCs) were 0.86–0.95. Section title: Measurements Educational score: 2.2647600173950195 Domain: biomedical Document type: Other Language: en 1. ACL attachment position of each bundle of BR‐VACLG and OR‐VACLG on CT Section title: Measurements Educational score: 4.1331634521484375 Domain: biomedical Document type: Study Language: en To identify the attachment positions of F‐AM and F‐PL, the medial femoral condyle was removed so that the lateral wall of the intercondylar notch could be observed from the medial side in line with the trans‐epicondylar axis. To identify the attachment positions of T‐AMM, T‐AML and T‐PL, a bird's eye view of the tibial plateau was obtained, and a line tangential to the posterior condyles of the tibial plateau was first determined as the mediolateral axis, followed by the perpendicular anteroposterior axis. The location of the attachment position of each bundle of BR‐VACLG and OR‐VACLG was evaluated using the quadrant method on these 3D images . The intra‐observer and inter‐observer ICCs of the ACL attachment position were 0.86 and 0.78, respectively. Section title: Measurements Educational score: 2.2060868740081787 Domain: biomedical Document type: Other Language: en 2. Comparison of orientation between the N‐ACL and the VACLG Section title: Measurements Educational score: 4.155305862426758 Domain: biomedical Document type: Study Language: en The angle between the ACL bundle and the tibial articular surface (ACL‐tibial plateau angle: ACL‐TP angle) was measured in oblique‐coronal and sagittal images based on the method reported by Take et al. . The tibial plateau was approximated by a plane in a scout view of frontal and lateral planes, which was defined as the axial plane. Next, the sagittal plane was defined perpendicular to the axial plane and the line tangential to the posterior edge of the tibial plateau. At the intercondylar level of sagittal slices, ACL grafts were clearly visible running obliquely beneath the intercondylar roof, or Blumensaat's line. The depicted ACL graft in the sagittal plane was then used to determine the oblique‐coronal plane, which contained a line tracing the centre of the ACL graft, and it was also set parallel to the line tangent to the posterior edge of the tibial plateau. The 3D orientation of the ACL bundle was represented by the ACL‐TP angle measured in the oblique‐coronal and sagittal planes. The ACL‐TP angles of the BR‐VACLG and OR‐VACLG were measured with the same slices used for the N‐ACL measurements. ACL‐TP angles of each bundle in the BR‐VACLG, OR‐VACLG, and N‐ACL were compared . The intra‐observer and inter‐observer ICCs of ACL‐TP angle measurements were 0.81 and 0.65, respectively. Section title: Statistical analysis Educational score: 3.977154493331909 Domain: biomedical Document type: Study Language: en Statistical analysis was performed using SPSS Statistics ver. 26 (IBM Corp.). The femoral attachment positions of each bundle of BR‐VACLG and OR‐VACLG by the Quadrant method were compared using the Wilcoxon signed‐rank test, with an alpha level of 0.05. Comparisons of the ACL‐TP angle of each bundle among N‐ACL, BR‐VACLG and OR‐VACLG were performed using the Friedman test with Bonferroni adjustment . Section title: RESULTS Educational score: 2.593506336212158 Domain: biomedical Document type: Study Language: en A total of 14 normal left knees (7 males and 7 females, mean age 27.8 ± 4.6 years) were included. Section title: RESULTS Educational score: 2.093312978744507 Domain: biomedical Document type: Other Language: en 1. Tunnel positions of each bundle based on the BLS Section title: RESULTS Educational score: 4.132347583770752 Domain: biomedical Document type: Study Language: en On BR‐VACLG, the femoral tunnels were located at 13.6 ± 2.5%/22.1 ± 9.0% for the F‐AM and 25.3 ± 3.1%/52.4 ± 7.0% for the F‐PL in deep‐shallow/high‐low directions by the quadrant coordinate system (Table 1 ). The femoral tunnels of OR‐VACLG were located at 24.9 ± 2.8%/30.5 ± 3.6% for the F‐AM and 37.2 ± 4.2%/59.3 ± 3.9% for the F‐PL in deep‐shallow/high‐low directions. Hence the femoral tunnels of the OR‐VACLG were statistically significantly shallower and lower compared to those of the BR‐VACLG in both F‐AM ( p = 0.012 and p = 0.036, respectively) and F‐PL ( p = 0.012 and p = 0.018, respectively). Section title: RESULTS Educational score: 4.145159721374512 Domain: biomedical Document type: Study Language: en The tibial tunnels were located at 44.0 ± 1.5%/23.4 ± 2.9% for the T‐AMM, 50.0 ± 1.9%/28.0 ± 2.9% for the T‐AML and 43.5 ± 1.6%/39.2 ± 3.0% for the T‐PL in medial‐lateral/anterior‐posterior directions (Table 2 ). Section title: RESULTS Educational score: 2.082598924636841 Domain: biomedical Document type: Study Language: en 2. Comparison in orientation among N‐ACL, BR‐VACLG and OR‐VACLG Section title: RESULTS Educational score: 4.11665678024292 Domain: biomedical Document type: Study Language: en ACL‐TP angles of the BR‐VACLG and N‐ACL were 75.2 ± 4.5° and 74.4 ± 3.4° for AMM, 82.9 ± 5.1° and 81.9 ± 3.8° for AML, 70.0 ± 7.2° and 71.1 ± 6.4° for PL in oblique‐coronal slices; 53.9 ± 4.4°and 55.3 ± 4.9° for AMM; 54.7 ± 2.6° and 54.9 ± 4.5° for AML; 51.2 ± 2.4° and 51.4 ± 3.3° for PL in oblique‐sagittal slices. There was no significant difference in the angles between N‐ACL and BR‐VACLG on the oblique‐coronal slices ( p = 0.617 for AMM, p = 0.261 for AML, and p = 0.617 for PL) or the oblique‐sagittal slices ( p = 0.211 for AMM, p = 0.453 for AML, and p = 0.901 for PL) . Section title: RESULTS Educational score: 4.166681289672852 Domain: biomedical Document type: Study Language: en The angles of the OR‐VACLG were 68.7 ± 5.0° for AMM, 76.3 ± 4.0° for AML, and 61.0 ± 4.7° for PL in the oblique‐coronal slice, and 50.5 ± 4.3° for AMM, 50.7 ± 3.2° for AML, and 48.1 ± 2.0° for PL in the oblique‐sagittal slices (Table 3 ). The angles of AMM and PL in OR‐VACLG were statistically significantly lower than those of N‐ACL on the oblique‐coronal slices ( p = 0.006 for AMM and p = 0.001 for PL) or the oblique‐sagittal slices ( p < 0.001 for AMM and p = 0.012 for PL), and those for all three bundles of OR‐VACLG were statistically significantly lower than those of BR‐VACLG on the oblique‐coronal slices (p = 0.001 for AMM, p < 0.001 for AML, and p = 0.006 for PL) or the oblique‐sagittal slices ( p = 0.018 for AMM, p = 0.003 for AML, and p = 0.018 for PL) . Section title: DISCUSSION Educational score: 4.1187639236450195 Domain: biomedical Document type: Study Language: en The most important finding of this study was that the BR‐VACLG showed a similar orientation to the N‐ACL on MRI in the normal fully extended knees. Comparison in ACL‐TP angles of each fibre bundle on both oblique‐coronal and sagittal slices between the BR‐VACLG on the CT model and the N‐ACL on MRI, showed no significant differences. In other words, each fibre bundle in the BR‐VACLG based on the BLS accurately mimicked the orientation of the N‐ACL, in contrast to that in the OR ‐ VACLG. Section title: DISCUSSION Educational score: 4.251274108886719 Domain: biomedical Document type: Study Language: en In anatomic ACL reconstructions, it could reasonably be assumed to mimic the normal ACL orientation by creating tunnels within the ACL attachment areas that have some BLs in their vicinity, as reported in the past. Iwahashi et al. reported that the ACL femoral attachment area was attached to the concave region behind the RR by histological evaluation . Ferretti et al. reported the presence of the lateral bifurcate ridge between the AM and PL bundles in the femoral attachment area . The ACL tibial attachment has been reported to have a C‐ or boot‐like shape . Tensho et al. reported that the ACL is attached to the posterior medial side of the bony prominence of the L‐shape formed by the bony landmarks AR and MIR . Kusano et al. reported that the CIR is located at the boundary between the ACL and the anterior horn of the lateral meniscus . The size of the attachment areas is reported as follows: 15–18 mm in length and 7–10 mm in width on the femoral side; 7–11 mm in length for AR, 10–13 mm in length for MIR, about 5 mm in length for CIR and about 5 mm of MIR‐CIR distance on the tibial side . Thus, in the present study, based on the BLS, two 7‐mm‐diameter circles straddling the lateral bifurcate ridge in the area behind the RR were used as the ACL femoral attachment points for the AM (AMM + AML) and PL bundles, respectively. Three circles, 5 mm in diameter, placed posterior to the AR, lateral to the MIR and medial to the CIR, were used as ACL tibial attachment points for the AMM, AML and PL bundles, respectively. Then, VACLG of the three lines connecting the centres of each fibre bundle could closely mimic the N‐ACL in the extended knee, showing that anatomic grafting in ACL reconstruction could be achieved by the BLS. Section title: DISCUSSION Educational score: 4.148060321807861 Domain: biomedical Document type: Study Language: en Clinically, Take et al. evaluated the tunnel position of ATB‐ACLR based on the BLS by the quadrant method. F‐AM was 13.2%: 21.1%, F‐PL 20.0%: 48.5% (deep‐shallow: high‐low directions) on the femoral side, while T‐AMM was 44.1%: 26.8%, T‐AML was 49.6%: 27.3% and T‐PL was 45.4%: 38.0% (medial‐lateral: anterior‐posterior directions) on the tibial side, which were comparable to the tunnel positions for BR‐VACLG in the present study . They also reported excellent clinical results in which the mean side‐to‐side difference of anterior laxity measured by the KT‐1000 arthrometer at one year after ATB‐ACLR was 0.4 ± 1.2 mm with the tibio‐femoral positional relationship normalised . Thus, the BLS could make it possible to reproducibly create anatomic tunnels leading to consistent restoration of stability, as proposed by Shino et al. . Section title: DISCUSSION Educational score: 4.092159748077393 Domain: biomedical Document type: Study Language: en The present study has some limitations. First, the sample size is small, so statistical power is reduced. Second, there were some cases in which the BLs were not clearly depicted on CT. It has been reported that 100% of the RR , 96%–100% of the AR and 100% of the MIR and CIR can be visualised on CT , but only about 80% of the lateral bifurcate ridge can be visualised by histological and arthroscopic examination . In the present study, the RR, AR, MIR and CIR were 100% delineable, but the lateral bifurcate ridge was unclear in 4 out of 26 knees (16%). However, the two 7‐mm‐diameter regular circles placed posterior to the RR in this study were considered properly positioned within the femoral attachment of the ACL, and the results would be similar even if the lateral bifurcate ridge were unclear. Third, the ACL‐TP angle was evaluated in a non‐weight‐bearing, static position. The CT and MRI images of the same knee had to be completely overlapped, so the scans were performed in extension under non‐weight‐bearing conditions. However, the conclusion drawn from this study may be different in the other conditions, as ACL tension/length is influenced by the knee flexion angle, weight‐bearing and quadriceps muscle contraction. Section title: CONCLUSION Educational score: 3.0081114768981934 Domain: biomedical Document type: Other Language: en The virtual triple‐bundle ACL graft on the 3D CT via the femoral tunnels behind the resident's ridge showed the equivalent orientation to the normal ACL on MRI. Section title: AUTHOR CONTRIBUTIONS Educational score: 0.9693721532821655 Domain: other Document type: Other Language: en Konsei Shino and Hiroyuki Yokoi conceived the hypothesis and designed this study. Hiroyuki Yokoi, Tomoki Ohori and Konsei Shino collected data. Narihiro Okazaki, Konsei Shino and Hiroyuki Yokoi analysed and interpreted the results and drafted the manuscript. Tomoki Ohori supported statistical analyses. All authors read and approved the final manuscript. Section title: CONFLICT OF INTEREST STATEMENT Educational score: 0.8662789463996887 Domain: other Document type: Other Language: en The authors declare no conflicts of interest. Section title: ETHICS STATEMENT Educational score: 1.0544626712799072 Domain: biomedical Document type: Other Language: en The research related to human use has been conducted in accordance with all the relevant national regulations and institutional policies and the tenets of the Helsinki Declaration, and it has been approved by the institutional review board of Yukioka Hospital. Informed consent has been obtained from all individuals included in this study. | Study | biomedical | en | 0.999997 |
PMC11696278 | Section title: Introduction Educational score: 4.162508964538574 Domain: biomedical Document type: Clinical case Language: en Enterogenous cysts (ECs) are rare, benign, congenital ectopic endodermal cysts that are lined by gastrointestinal or respiratory mucin-secreting epithelium. ECs typically occur in the mediastinum and abdomen, and are infrequently found in the central nervous system (CNS) ( 1 ). In the CNS, ECs are usually located extramedullary in the spinal cord and rarely located intracranially, which are commonly found in the cerebellopontine cistern, pre-pontine cistern, or ventriculus quartus cerebri ( 2 ). ECs occur more often in male adolescents, with a male-to-female ratio of 3:1 to 2:1 ( 2 ). Here, we reported an extremely rare case of EC that was located in the brainstem of a 17-year-old male adolescent. Before that, only seven reports have described brainstem ECs in pediatric patients (0–18 years) ( Table 1 ). Among those cases, only four patients had achieved tumor total resection, and our case has the largest brainstem EC that had been completely resected. Section title: History and imaging Educational score: 3.916452169418335 Domain: clinical Document type: Clinical case Language: en A 17-year-old male patient presented in our outpatient with 6 months of dizziness and 12 months of right limb weakness, which was aggravated and resulted in the inability to flex the right lower limb and unsteady walking for 1 month. The patient, without any family history or medical history, was diagnosed by the local hospital as having a brainstem tumor and was transferred to our hospital. The physical and neurological examinations showed that the muscle strength of his limbs was class V on the left side, class IV in the extensors, and class II in the flexors on the right side. Apart from these, no other positive signs were detected in the examinations. Head computed tomography (CT) scanning showed a cauliflower-like hyperdense lesion located in the brainstem . On magnetic resonance imaging (MRI), the lesion was extracerebral and located in the ventral side of the brainstem, compressing the brainstem evidently. The lesion was mostly hyperintense on T1- and T2-weighted scanning, with prominent gadolinium enhancement . Moreover, there was a small nodule in the right part of the lesion that showed a low signal on T1, T2, and enhanced scanning, suggesting a calcification within the lesion. The lesion measured approximately 47 × 38 × 36 mm in size, which severely compressed the brainstem, causing it nearly to be a straight line with only 3 mm at the narrowest point . Based on the above clinical evidence, the patient was preoperatively diagnosed as having an epidermoid cyst. Because of the evident clinical symptoms and prominent mass effect on the brainstem, we decided to operate on this patient. Section title: Surgical details and histopathology Educational score: 3.909118413925171 Domain: clinical Document type: Clinical case Language: en The patient was positioned laterally and surgery was performed using a far lateral approach, craniotomy with an extension from the occipital midline through the foramen magnum to the left condyle of occipital bone, ending in the star point covering the transverse sinus. After partially removing the occipital condyles, incising the posterior arch of the atlanto-axial, and cutting the endocranium, we explored the left ventral side of the cerebellum using a microscope. The lesion was exposed in the ventral part of the pons, which was embedded in the pons and severely compressed, displaced, and deformed it. The mass was cystic, with an intact outer envelope, and a thick yellowish cystic liquid content. Fortunately, the cyst wall did not adhere tightly to the brainstem. Therefore, after aspirating the cyst fluid, we carefully stripped the cyst wall from the brainstem and removed the cyst completely. Section title: Surgical details and histopathology Educational score: 3.8672420978546143 Domain: clinical Document type: Clinical case Language: en Microscopically, the cyst wall was covered with pseudo-stratified epithelium with abundant foam cells around. Under the epithelium is fibrous tissue, with collagen, as well as little capillaries between tissues . In the cyst fluid, there were a large number of erythrocytes and a small number of leukocytes. Postoperatively, CT and MRI showed that the cyst was completely removed . After the postoperative treatment including lumbar puncture, postoperative limb function exercise, and neurotrophic drug administration, the patient’s symptoms have partially improved with a muscle strength of class IV in the flexors on the right side, compared with that of class II before the surgery. The patient and his parents were very satisfied with the treatment. There was no tumor progression over the next 6 months of follow-up, nor were there any recurrences. When he came back 6 months later, his symptoms have totally improved with a muscle strength of class V on the limbs. Section title: Discussion Educational score: 4.3568034172058105 Domain: biomedical Document type: Study Language: en ECs are defined as benign cystic lesions lined by gastrointestinal or respiratory mucin-secreting epithelium, which were first found by Puussepp in 1934, but were officially named enterogenous cysts in 1958 by Harriman ( 10 , 11 ). The pathogenesis of ECs is still controversial, which most researchers believed should be ascribed to the mutation or hypoplasia of the ectoblast and entoderm during the third embryonic week ( 2 ). From the beginning of the third embryonic week, the ectoblast, gradually developing with neural tubes, and the entoderm, differentiating into the intestinal tube, will divide from the ectoblast along with the embryonic development. In the case of impaired, vestigial, or ectopic embryo development, EC will form, which is usually combined with gastrointestinal, spinal, or medullispinal malformations ( 2 ). ECs can occur at any age, with more prevalence in male patients ( 2 , 12 ). However, ECs of male patients are more likely to be found in the spine, and those of female patients are more likely to be found intracranially ( 12 ). ECs of CNS mostly occur in the spinal canal (more than 80%), accounting for 0.3% to 0.5% of the intraspinal tumor, with a higher prevalence in the cervical and upper thoracic segments, which preferably occur in the ventral part of the subdural spinal cord ( 12 ). In our case, the patient was a male adolescent, who was found to have an EC in a very rare location of the brainstem without any other intracranial malformations. Until now, there have been only seven cases of brainstem ECs reported in pediatric patients. Among these cases, only four had achieved total tumor resection, and the tumor in our case is the largest in size among the completely resected tumors. Section title: Discussion Educational score: 3.4297871589660645 Domain: biomedical Document type: Other Language: en The clinical manifestations of ECs are closely related to the pathogenic site, with long-term recurrent headache being the first symptom in most cases. Later on, along with cyst enlargement, the mass effects gradually appear, causing epilepsy, intracranial hypertension, and dysneuria. Even more, aseptic meningitis may occur in case of a fistula formation. Section title: Discussion Educational score: 4.243291854858398 Domain: biomedical Document type: Study Language: en The diagnosis of intracranial ECs relies mainly on imaging and pathology. Because of the slow growth of cysts, low incidence, variable clinical symptoms, and atypical imaging manifestations, it is difficult to distinguish them from other intracranial mass lesions, which leads to a difficult preoperative diagnosis. CT scanning can only manifest the location of the lesion and the feature of cystic changes. MRI scanning can distinctly show the shape of the lesion and its relationship to the surrounding brain tissue. On MRI scanning, most ECs of CNS have thin, uniform walls, and smooth margins, with little edema in the surrounding brain tissue. On T1- and T2-weighted scanning, the cyst usually displays a signal equal to or slightly higher than that of the cerebrospinal fluid (CSF), with no enhancement or slight enhancement in case of fibrillation of partial cyst wall on enhanced scanning ( 10 , 13 ). In our case, the lesion displayed marked hyperintensity on both T1- and T2-weighed scanning with prominent gadolinium enhancement, which is not in accordance with the reported cases. This nonconformity of signals in MRI may be attributed to the high protein content in cystic fluid. Section title: Discussion Educational score: 4.1725358963012695 Domain: biomedical Document type: Study Language: en Histologically, ECs can be classified into three groups based on the histological origin of cyst epithelium. Group I: The cyst walls are lined by a monolayer, pseudocompound cubic or columnar epithelium, with or without fiber hairs. Group II: The cysts, in addition to the above structure, may be composed of mucous glands, plasma glands, and ganglia. Group III: The cysts, in addition to the findings in group II, may be composed of ventricular membrane and neuroglial components ( 14 ). In immunohistochemical staining, EC cells show positive expression of epithelial membrane antigen and carcinoembryonic antigen, with negative expression of glial fibrillary acidic protein (GFAP) ( 15 ). Section title: Discussion Educational score: 4.157504081726074 Domain: biomedical Document type: Study Language: en As a congenital disease, ECs should be distinguished from intracranial dermoid cysts, epidermoid cysts, and arachnoid cysts. Dermoid cysts are usually seen in adults (at least 40 years old), with the sellar region or cranial fossa being the common location ( 16 ). On account of their fatty component, dermoid cysts display heterogeneous signals on MRI scanning, which often shows a slight hypointense signal or an occasionally hyperintense signal on T1-weighted scanning, and a slight hyperintense signal on T2-weighted scanning, without gadolinium enhancement. The imaging displays of epidermoid cysts are multifarious, depending on the composition and proportion of the cysts, which usually show a hypointense signal (slightly higher than the CSF signal) on T1-weighted scanning and a hyperintense signal on T2-weighted scanning ( 17 ). The arachnoid cysts display signals similar to the CSF, which are hypointense signals on T1-weighted and hyperintense signals on T2-weighted scanning. In addition, the expression of GFAP can be used for differential diagnosis from ECs and arachnoid cysts. Section title: Discussion Educational score: 4.02644681930542 Domain: biomedical Document type: Study Language: en The treatment of ECs is based on surgical resection, with the goal of complete removal of the lesion. Even if the cyst is completely removed, it may still recur, worsening the symptoms of neurological deficit and increasing the risk and difficulty of reoperation ( 18 – 20 ). Here, we present several operative experiences: (1) To avoid aseptic inflammation, tampons should be applied around the cyst during excision to prevent leakage of cystic fluid, and the operative region should also be repeatedly irrigated after the resection. (2) During the operation, we found that the cyst has severely compressed the brainstem, significantly deforming it. However, the cyst wall was not tightly adherent to the brainstem, which resulted in the complete excision of the cyst. Nevertheless, we still do not recommend total resection of brainstem ECs at the expense of damage to the surrounding tissues, especially the brainstem. With regard to the cyst being tightly adherent to the brainstem, we suggest that partial resection should be performed first, followed by electrocautery of the remaining part of the cyst wall. (3) Although ECs are considered as benign lesions, there have already been reports about cases with recurrence, dissemination, and canceration ( 18 , 19 ). Therefore, intraoperative freezing pathological examination is proposed, which plays an important role in the selection of the operative approach, the resection scope, and the prognosis of intracranial ECs. Section title: Conclusion Educational score: 4.054300308227539 Domain: biomedical Document type: Clinical case Language: en We present a complete surgical resection of a rare huge brainstem EC, in which total resection had been reported in only four cases. Intracranial ECs are congenital benign lesions, the diagnosis of which fundamentally relies on imaging and histological results. Signals of ECs on MRI are multiple, depending on the content of the cyst. Typically, the pathological characteristic of ECs is the presence of cyst walls lined by a monolayer, pseudocompound cubic or columnar epithelium, which can be further classified into three groups. Surgical resection is still the primary treatment for intracranial ECs, since radiotherapy and chemotherapy have not been proven to have a therapeutic effect. Therefore, further investigations are needed to optimize management and recurrence avoidance. | Clinical case | biomedical | en | 0.999999 |
PMC11696280 | Section title: Introduction Educational score: 1.4317266941070557 Domain: other Document type: Other Language: en Colourism refers to prejudice in which people are penalised the darker their skin is and the further their features are from those associated with whiteness. Intrinsically related to, and yet distinct from racism (i.e., prejudice and discrimination based on racialised), colourism occurs both within and across racialised groups . When colourism is experienced from a different racialised group, it can intensify experiences of racism, such that those with darker skin are more likely to experience racialised discrimination . In this way, colourism and racism operate together . However, within a racialised group, the prejudice operates in the absence of racism as a skin shade (and features) hierarchy in favour of those with lighter skin and phenotypical features that are associated with whiteness. Section title: Introduction Educational score: 1.095566749572754 Domain: other Document type: Other Language: en Colourism has a widespread impact on the lives of People of Colour 1 globally and can affect many different aspects of an individual’s life. While people with light skin often benefit from light-skin privilege, those with dark skin experience penalties in numerous institutional settings, including in education, health and the criminal justice system . Colourism also can affect experiences in the relationship market, particularly for women . As a racialised and gendered phenomenon, colourism can affect people differently according to their ethnicity and gender . Consequently, it is valuable to take an intersectional approach that is attentive to how colourism is perpetuated, reproduced and experienced at the individual, interpersonal, and systemic level in relation to the interplay of different personal characteristics, such as skin shade, ethnicity and gender. In this article, we focus on the experiences of Black and mixed Black-White women in recognition of the ways in which colourism compounds experiences of anti-Black racism. 2 Section title: Introduction Educational score: 0.9226894974708557 Domain: other Document type: Other Language: en Colourism is pervasive and it is sustained and reproduced in multiple ways through powerful sociocultural influences including family, peers, and the media . In this article, we focus on how the media can serve to popularise, normalise, and perpetuate colourist ideas through the ways in which it both depicts and excludes minoritised ethnic people, and particularly women with dark skin . The media, including film, TV, advertising and print media (e.g., newspapers and magazines), plays a potent role in contributing to wider colourist attitudes that can be internalised by individuals and reinforced by family and peers. Section title: Introduction Educational score: 1.0274567604064941 Domain: other Document type: Other Language: en Operating alongside racism, colourism informs dominant media-sustained appearance ideals for women, which serve to privilege whiteness and light skin as attractive and desirable . For example, Keigan et al. argue that “Eurocentric physical characteristics,” such as thinness, whiteness and long, straight hair are portrayed as the epitome of beauty” in the media. By positioning women with White or light skin as beautiful and desirable, women with dark skin are implicitly positioned as lacking in both beauty and desirability by the media and society more broadly . For example, Collins argues that “controlling images,” which are designed to make forms of social injustice, such as racism, sexism, and colourism, “appear to be natural, normal, and inevitable parts of everyday life,” construct Black women as “ugly and unfeminine” (p. 27). Section title: Introduction Educational score: 1.2115145921707153 Domain: other Document type: Other Language: en Due to “patriarchal patterns of desire” , women and men’s experiences of colourism can differ, as women are typically judged on their looks more than men are, subjected to more societal appearance pressure, and held to different appearance ideals where light skin is consistently privileged . Additionally, as dark skin is often associated with masculinity and virility, Black men, unlike Black women, can benefit from having darker skin in the context of societal appearance ideals and romantic relationships . Section title: Introduction Educational score: 1.2080821990966797 Domain: other Document type: Other Language: en Colourism can inform heterosexual men of colour’s choice of romantic partners, and their descriptions of the physical attributes that make women attractive . For example, Hunter found that when it came to the marriage market, Black women in the USA who had light skin “had a clear advantage” over those with darker skin and were more likely to marry men of high-status. Experimental research from the US showed that Black men rated the same set of advertising images featuring Black women as significantly more attractive when their skin was digitally lightened compared with when their skin was digitally darkened . Black men in Phoenix and Craddock’s UK study explained that one reason why some Black men prefer women with lighter skin is due to the elevated social status they can gain from having a partner with light-skin privilege. Section title: Colourism in the UK Educational score: 1.2339531183242798 Domain: other Document type: Study Language: en Colourism is under-researched in the UK. However, a few studies have been published in the past few years that give some insights into how the prejudice operates in the UK. For example, in a cross-sectional, mixed ethnicity study on colourism, Craddock et al. found that compared with people from other minoritised ethnic groups, Black people reported greater experiences of colourism. Further, those with darker skin shades reported greater experiences of colourism. In turn, colourism was associated with negative wellbeing outcomes even when racism was included in the model, thus highlighting the unique role of colourism. Notably, qualitative work conducted in the UK that has explored the effects of colourism and racism has shown that in predominantly White areas, people of mixed ethnic backgrounds and Black people with light skin may be subjected to anti-black racism and may not experience the privileges they may otherwise experience in more diverse contexts . Section title: The present work Educational score: 1.219690203666687 Domain: other Document type: Study Language: en In this paper, we analyse narratives from Black and mixed Black-White women and men in an exploration of how skin shade affects Black and mixed Black-White women’s social positioning in the context of romantic relationships. Specifically, we take an intersectional approach focusing on ethnicity, gender and skin shade to explore the complex processes through which colourism positions Black or mixed Black-White women who have dark or light skin in hierarchies of beauty and desirability . We consider the implications of this in terms of both how women are positioned in the heterosexual relationship market, and how it affects relationships between women with dark and light skin. Section title: The present work Educational score: 1.0648016929626465 Domain: other Document type: Other Language: en Our work is situated in Britain (which includes England, Scotland, and Wales). Britain is majority White ranging from 81.7% White in England and Wales, to 87.1% White “Scottish” in Scotland ( Office for National Statistics (ONS), 2022 ; National Statistics, 2024 ). The Black population (categorised as Black, Black British, Caribbean or African) is 4.0% in England and Wales and 1.0% in Scotland, and the mixed Black-White population is 1.1% in England and Wales. 3 Britain has a long history of racism, including in its institutions . Accordingly, our work examines colourism against a backdrop of racism in Britain. Section title: Participants Educational score: 1.9224525690078735 Domain: biomedical Document type: Study Language: en Participants were 27 adults (18 women, 9 men) racialised as Black ( n = 18) or mixed Black-White ( n = 9) living in Britain. 4 The mean age was 35.8 years, ranging from 19 to 60 years old. Six participants (five women and one man) were full-time students (undergraduate or postgraduate), and one woman was a stay-at-home parent at the time of interview. The remaining participants worked in a variety of occupations in sectors including government, healthcare, media, and transport. All participants identified as heterosexual, except for one woman who identified as pansexual. Demographic data pertaining to social class or social economic status was not collected. Participant demographics, self-reported skin shade, and allocated pseudonyms are detailed in Table 1 . Section title: Skin shade chart Educational score: 2.0448102951049805 Domain: biomedical Document type: Study Language: en We created a purpose-built skin shade chart using 30 colours from Pantone® SkinTone™ Guide to use as an ice-breaker, and to capture participants’ skin shade as they saw it. The Pantone® SkinTone™ Guide is designed to be representative of the full spectrum of human skin types and in 2019 contained 110 SkinTone™ Guide shades. We selected 30 shades that we felt adequately represented minoritised ethnic people from different racialised groups and that were sufficiently distinct when printed on a home printer. We felt that 30 shades gave enough range without being overwhelming to look at in a brief exchange at the start of the interview. Section title: Interview guide Educational score: 1.797629475593567 Domain: other Document type: Study Language: en We followed a semi-structured interview schedule that we designed for the scoping research project. The semi-structured interview format provided flexibility, allowing the conversation between the interviewer and participant to delve into areas that felt most relevant to the participants, in such a way that data were co-constructed. Interview questions were intentionally broad to gain initial insights into the topic of colourism in Britain. For example, questions included: How do you think others view your skin shade? Do you feel pressure to change the shade of your skin? Have you ever received preferential treatment because of the shade of your skin? Have you ever been discriminated against or treated badly because of the shade of your skin? Section title: Procedure Educational score: 1.7278519868850708 Domain: other Document type: Study Language: en Ethical approval for a broad, scoping qualitative research project on colourism and skin shade in the UK was granted by the Research Ethics Committee at SOAS, University of London. Study advertisements were shared on social media inviting minoritised ethnic people (aged 18 and over) living in the UK to take part in a 60-min interview on their experiences related to their skin shade. Snowball sampling was also used. Interested individuals were sent a study information sheet and consent form. There were no incentives for participation. Section title: Procedure Educational score: 1.9730596542358398 Domain: biomedical Document type: Study Language: en Semi-structured interviews took place in person or via video conferencing software. The first author conducted 18 interviews, and the second author conducted nine. Demographic information was collected before formally starting the interview, with participants either completing a short questionnaire or answering demographic questions verbally. Then, we asked participants to indicate the colour that was the closest match to their skin shade on the chart before we proceeded with the interview, using our schedule. Section title: Procedure Educational score: 1.796221137046814 Domain: other Document type: Study Language: en Interviews were audio recorded with participants’ consent and were then transcribed by either a professional transcription service, freelance transcribers, or one of the study authors. To protect participants’ privacy, pseudonyms were used and identifying information was removed from participant accounts. Audio recordings were deleted once transcripts were checked by one of the study authors. Section title: Data analysis Educational score: 1.9813646078109741 Domain: biomedical Document type: Study Language: en The first author led the data analysis using reflexive thematic analysis . This approach was selected as it is a theoretically flexible approach, suitable for under-researched topics. Section title: Data analysis Educational score: 1.3552335500717163 Domain: other Document type: Other Language: en Analysis was informed by both social constructivism and intersectionality. Socio-constructivism posits that reality is socially constructed through interactions and shared understandings among individuals . There is no single, objective reality independent of human perception and social context. Reality is seen as constructed through language, culture, and social practices. Socio-constructivism emphasises the role of human agency in creating and interpreting the world . Section title: Data analysis Educational score: 3.013684034347534 Domain: other Document type: Study Language: en Adding an intersectionality framework allowed us to understand the nuanced and multifaceted experiences of participants in relation to their personal characteristics, with a particular focus on gender, minoritised ethnic group and skin shade. Intersectionality, a concept coined by Crenshaw , emphasises the interconnectedness of social identities such as “race,” gender, age, class, and sexuality, and how these intersections create unique modes of discrimination and privilege. By acknowledging that individuals’ experiences cannot be fully understood through single-axis analyses, this approach allows for a more comprehensive exploration of how various forms of inequality and identity overlap and interact . Further, intersectionality provides a lens through which to examine the complexity of participants’ lived experiences and the structural contexts that shape them . By drawing on this framework, the study aims to capture the diverse and intersecting factors that influence participants’ understandings and experiences of colourism in the context of desire and relationships. However, it should be noted that our intersectional analyses are most attentive to gender, ethnicity and skin shade based on a predominantly heterosexual sample. Interpretation based on age and social class were beyond the scope of the present work as our sample was not large enough to draw meaningful comparisons and interpretation. Section title: Data analysis Educational score: 3.7145495414733887 Domain: other Document type: Study Language: en The six-stage guidance for reflexive thematic analysis detailed by Braun and Clarke was followed: (i) data familiarization, (ii) generating initial codes, (iii) generating themes, (iv) reviewing potential themes, (v) defining and naming themes, and (vi) writing up the results. Codes were generated in a two-step process. First, interviews were coded openly, guided by participants’ meanings. Then, a second round of deductive analysis was conducted, drawing on existing knowledge and understandings of colourism. The authors had several discussions about the data and candidate themes. The second author also provided feedback and revisions on the defining and naming of themes and the write up. Section title: Positionality and reflexivity Educational score: 0.9563857913017273 Domain: other Document type: Other Language: en Both authors are minoritised ethnic women. The first author is an African Caribbean sociologist. The second author has an educational background in psychology and is of mixed ethnicity, with one Indian and one White parent. We both have experience of using qualitative methods and interviewing individuals on sensitive and personal topics. Section title: Positionality and reflexivity Educational score: 1.4525808095932007 Domain: other Document type: Study Language: en We did not seek to match participants’ broad racialised group when we assigned an interviewer as we were interested in exploring participants’ experiences as both “insiders” and “outsiders.” As an African Caribbean woman with dark or medium-dark skin, the first author had an insider status when interviewing Black women, some of whom remarked on how easy it was to talk to her. However, it may have been more awkward for mixed Black-White women to discuss the tensions they felt with Black women in conversation with a Black woman researcher. In contrast, the second author occupied insider status with participants of mixed ethnicity who all similarly had one White parent, which fostered some shared understanding in racialised dynamics and feelings of belonging. The second author’s outsider status in terms of racialised group may have been useful as it may have prompted more explanation and description, however, it may have limited the extent to which participants’ felt comfortable sharing. Section title: Positionality and reflexivity Educational score: 1.3140754699707031 Domain: other Document type: Study Language: en As two women, we could not match interviewer-participant by gender for the men in our study. Prior to data collection, we wondered if men may be reticent about openly expressing their feelings on a sensitive topic that includes gendered dynamics. In fact, we found that men were forthcoming in their narratives. However, we do not know how they would have responded had they been speaking with a man, and particularly with one from the same racialised group as themselves. Section title: Results Educational score: 1.123004674911499 Domain: other Document type: Study Language: en Our results are presented in four sections that consider the implications of different aspects of colourism for Black women with dark skin and Black and mixed Black-White women with light skin. The first explores the effect of colourist appearance ideals and colourism in the media. The second examines the impact of internalised colourism informing heterosexual relationship choices. The third analyses how sexual colourism causes competition and tensions between women. The fourth section considers Black participants’ small acts of resistance when confronted with colourist, societal appearance ideals. Section title: “The media portrays an image that tells Black people … you are not beautiful”: mainstream media promoting colourist appearance ideals Educational score: 0.9207763075828552 Domain: other Document type: Other Language: en In this first section, we begin by exploring women’s perceptions of how racist and colourist “European standards of beauty” promoted by the media contribute to constructions of Black women as inferior and unattractive. We then examine narratives that argue that women with very dark skin and natural hair are underrepresented or negatively represented in the media, which can have adverse psychological effects and contribute to Black women feeling undesirable. Next, we analyse narratives from Black men who argue that the media promotes the interests of White people, portraying Black women in negative ways, which can lead them to feel unattractive. Section title: “The media portrays an image that tells Black people … you are not beautiful”: mainstream media promoting colourist appearance ideals Educational score: 1.0182701349258423 Domain: other Document type: Other Language: en Many participants pointed to the media to explain why Black women with dark skin and Afro hair are positioned at the bottom of what Hunter refers to as the ‘beauty queue.’ The term describes how women are ranked by beauty (defined in large part by racialisation and skin shade) in marriage and dating markets, where “beauty” operates as a form of racialised social or symbolic capital. For instance, Kelly, a Millennial who described her skin shade as “Galaxy milk chocolate,” said: Section title: “The media portrays an image that tells Black people … you are not beautiful”: mainstream media promoting colourist appearance ideals Educational score: 1.0087497234344482 Domain: other Document type: Other Language: en Kelly suggested that dominant perceptions of Black women are that they are inferior (“less than”) and unattractive with unpleasant hair. Consistent with scholars such as Keigan et al. , Kelly linked this to racist and colourist “European standards of beauty” in the media that privilege White or light skin and straight European hair. Section title: “The media portrays an image that tells Black people … you are not beautiful”: mainstream media promoting colourist appearance ideals Educational score: 1.1971251964569092 Domain: other Document type: Other Language: en In keeping with findings from a content analysis in the US, which found that Black women with dark skin were often excluded in media advertising , Alice, another Millennial, who had lighter skin, highlighted the lack of representation of Black and Asian women with “really dark” skin in the media. Section title: “The media portrays an image that tells Black people … you are not beautiful”: mainstream media promoting colourist appearance ideals Educational score: 0.993459939956665 Domain: other Document type: Other Language: en Her comment that “if you do not fit that mould … it does feed into your head,” suggests that there are psychological consequences of being excluded from the limited racialised constructions of beauty in the media. In addition to a lack of women with very dark skin and natural hair in the media, Alice said there is a “very narrow idea of what’s normal and what’s good,” indicating how morality is implicitly tied into media appearance ideals; that dark skin and Afro hair is neither “normal” nor “good.” This is linked to what Hunter described as “a colour-based halo effect”: that light skin is not only associated with beauty but also virtue and other positive qualities. Section title: “The media portrays an image that tells Black people … you are not beautiful”: mainstream media promoting colourist appearance ideals Educational score: 0.9816113114356995 Domain: other Document type: Other Language: en It was not just Millennial women who spoke about the negative impact of racialised and colourist appearance ideals for women. Portia, who belonged to Generation X and described her skin as “beautiful dusky brown,” said she used to feel “too dark, not nice” and highlighted the media as one of the sources of that negative self-perception. She said: Section title: “The media portrays an image that tells Black people … you are not beautiful”: mainstream media promoting colourist appearance ideals Educational score: 0.9883678555488586 Domain: other Document type: Other Language: en Her narrative suggests that while the media plays a role in indoctrinating people with colourist ideas, colourist messaging is ubiquitous (“everywhere”). Portia also highlighted a gender disparity in the representation of Black people in the media in relation to skin shade. She said that “[d]arker-skin Black men […] they are viewed as attractive, whereas, I think for the most part, dark-skinned Black women probably still are not, if you look at the television and/or that kind of industry.” Section title: “The media portrays an image that tells Black people … you are not beautiful”: mainstream media promoting colourist appearance ideals Educational score: 1.083900809288025 Domain: other Document type: Other Language: en Black men in our study also discussed the negative impact of racist and colourist gendered appearance ideals on Black people, and Black women in particular. For example, Ekow, a Millennial, said that the media can also have a damaging effect on Black people’s self-esteem through the images it conveys about beauty. He stated: Section title: “The media portrays an image that tells Black people … you are not beautiful”: mainstream media promoting colourist appearance ideals Educational score: 0.9443000555038452 Domain: other Document type: Other Language: en What he described is what Collins terms “controlling images.” Collins argues that some Black women internalise controlling images that construct them as “ugly and unfeminine” and “come to believe that they are the stereotypes.” To buffer this negative influence from the media, Ekow suggested that young Black people, and particularly women, require resilience and a supportive community to bolster their identity in order to feel beautiful and maintain high self-esteem. His comments echo those of an earlier text from Collins, published almost 30 years ago , who argued that “[e]nduring the frequent assaults of controlling images requires considerable inner strength.” Section title: “The media portrays an image that tells Black people … you are not beautiful”: mainstream media promoting colourist appearance ideals Educational score: 1.1159275770187378 Domain: other Document type: Other Language: en Further, several men in our study also emphasised the role the media, or more specifically White people who are in positions of power in the media, plays in placing this beauty burden on Black women. For example, Malakai, a Millennial with dark skin, said: Section title: “The media portrays an image that tells Black people … you are not beautiful”: mainstream media promoting colourist appearance ideals Educational score: 0.9767599105834961 Domain: other Document type: Other Language: en Relatedly, Bilal, a Millennial, who had a similar skin shade to Kelly, said: Section title: “The media portrays an image that tells Black people … you are not beautiful”: mainstream media promoting colourist appearance ideals Educational score: 0.9654267430305481 Domain: other Document type: Other Language: en In their narratives, Malakai and Bilal argued that the media shapes societal perceptions of Black beauty because those in positions of power are White and favour light skin and straight, smooth hair. Finally, Ekow, also a Millennial, argued that even when the media includes Black women, it prioritises those with features that can be associated with whiteness. Using the example of the Black supermodel, Naomi Campbell, he said: Section title: “The media portrays an image that tells Black people … you are not beautiful”: mainstream media promoting colourist appearance ideals Educational score: 0.9982738494873047 Domain: other Document type: Other Language: en The implication of Ekow’s argument is that colourism in the media is apparent even when women with darker skin shades are depicted because beyond their skin shade, it is those who fit White appearance ideals in other ways (e.g., thin, straight hair, a slim nose) who are included. This is consistent with Laybourn , who argued that White appearance ideals are maintained by “placing white beauty ideals onto black bodies.” Moreover, his comments resonate with historical perspectives in the media, such as the 1976 description of the supermodel Iman as “a White woman dipped in chocolate” from then editor-in-chief of Essence, Marcia Gillespie . Section title: “The only dark-skin people we like are the dark-skin men”: colourism shaping women’s experiences of romantic relationships Educational score: 0.9042263031005859 Domain: other Document type: Other Language: en We begin this section with an exploration of how, due to gendered colourism, Black men with dark skin are sought after in the relationship market, while women with dark skin are not. We suggest that Black men’s desirability means that those who have internalised colourism are well-positioned to perpetuate it in their relationship choices. We then complicate this by discussing how men can feel constrained in terms of their dating options due to concerns about other people’s colourist judgements. We conclude the section with an exploration of the issues that sexual colourism raises, including: the pain it can cause Black women with dark skin and the discomfort it can cause those with light skin. Section title: “The only dark-skin people we like are the dark-skin men”: colourism shaping women’s experiences of romantic relationships Educational score: 1.1470552682876587 Domain: other Document type: Other Language: en Participants frequently discussed in-group colourism, giving examples of Black people’s prejudicial attitudes and opinions about other Black people based on skin shade. This most commonly arose when speaking about heterosexual relationships where Black and mixed Black-White women with light skin were positioned as the most desirable by Black men, while Black women with dark skin were often overlooked. In contrast, Black men with dark skin were viewed as desirable. This phenomenon can be conceptualised as sexual colourism, drawing on the concept of “sexual racism” , in which people discriminate between possible romantic or sexual partners based on skin shade. Section title: “The only dark-skin people we like are the dark-skin men”: colourism shaping women’s experiences of romantic relationships Educational score: 1.0205856561660767 Domain: other Document type: Other Language: en Sofia and Bilal were among the participants who highlighted gendered in-group colourism, arguing that Black women with dark skin are viewed negatively, while Black men with dark skin are desired. Sofia, who belonged to Generation Z and described her skin as “light golden,” explained: Section title: “The only dark-skin people we like are the dark-skin men”: colourism shaping women’s experiences of romantic relationships Educational score: 0.9918827414512634 Domain: other Document type: Other Language: en In contrast to the association between femininity and light skin , Sofia suggested that women with dark skin are seen as “aggressive” by other Black people, which is a long-established trope. Assertive Black women are labelled “aggressive” and seen as “threatening” as they “challenge White patriarchal definitions of femininity” . While Black men with dark skin can be seen as aggressive and threatening too, they are also desired and Sofia said, they are perceived to be “the things to have.” In commenting that many Black men will disparage (“trash”) women of the same skin shade as themselves and their mothers, she highlights how deeply entrenched gendered colourism can be. Section title: “The only dark-skin people we like are the dark-skin men”: colourism shaping women’s experiences of romantic relationships Educational score: 0.9724130034446716 Domain: other Document type: Other Language: en Like Sofia, Bilal described how Black men’s experiences are distinct from women’s because they are sought after in the relationship market. He said: Section title: “The only dark-skin people we like are the dark-skin men”: colourism shaping women’s experiences of romantic relationships Educational score: 1.0698251724243164 Domain: other Document type: Other Language: en As Robinson argues, in terms of heterosexual relationships, Black men benefit from more options for relationships with people of other ethnicities than Black women do. For Black women with dark skin, colourism compounds the ‘gendered racism’ or ‘racialised sexism’ to which they are subjected . Section title: “The only dark-skin people we like are the dark-skin men”: colourism shaping women’s experiences of romantic relationships Educational score: 1.011813998222351 Domain: other Document type: Other Language: en The desirability of Black men with dark skin means that those who internalise colourist, gendered appearance ideals are likely to be well positioned to be able to perpetuate it in the relationship market. Using the example of his Black heterosexual male friends’ colourist dating preferences, Ekow said: Section title: “The only dark-skin people we like are the dark-skin men”: colourism shaping women’s experiences of romantic relationships Educational score: 0.9380673766136169 Domain: other Document type: Other Language: en Through stating that “on a subconscious level” Black men have been “programmed” to view Black women, and particularly Black women with darker skin, negatively, Ekow’s comment emphasises how colourist, gendered appearance ideals can be internalised. The fact that both Sofia and Ekow argue that some Black men shun women with similar skin shades to their mothers, emphasises both the complexity and perniciousness of internalised colourism . Section title: “The only dark-skin people we like are the dark-skin men”: colourism shaping women’s experiences of romantic relationships Educational score: 1.1295082569122314 Domain: other Document type: Other Language: en Several Black men in our study provided further nuance to how colourism shapes Black men’s dating preferences by highlighting the impact of concerns that other people might be colourist. For example, Isaiah, a Millennial who described his skin shade as light brown, said he was single and that he would “subconsciously be drawn towards females that are of a similar shade to myself.” When discussing appearance ideals for women, he said, “the ideal has a lot of external factors involved in it.” While his ideal woman would have very dark skin, societal racism and colourism mean that having a partner with dark skin would make him “uncomfortable” and self-conscious when out in public. He gave a hypothetical example: Section title: “The only dark-skin people we like are the dark-skin men”: colourism shaping women’s experiences of romantic relationships Educational score: 1.022074818611145 Domain: other Document type: Other Language: en His concern that a partner experiencing colourism would make him “uncomfortable” due to the injustice of it, meant that Isaiah said he would choose women with light skin instead. This suggests that societal colourism impacts men in complex ways, affecting not only who they desire, but who they are comfortable to be seen with. Relatedly, Ekow said that his first girlfriend was from South Sudan, and had dark skin, which used to be his preference, and “I knew some people that would make fun of that, like that, because she was dark skinned.” He said that now, “if I see someone that I like, I will ask myself, do I like this person just because they are light skinned? Or am I giving them more preference because they are light skinned? Because I’m aware that that happens.” This raises the question of whether his change in preference was related to the mocking he experienced while in his first relationship. These narratives suggest that concerns about the implications, and potential stigma, of being seen with women with dark skin can inform Black men’s dating preferences. Section title: “The only dark-skin people we like are the dark-skin men”: colourism shaping women’s experiences of romantic relationships Educational score: 1.0711604356765747 Domain: other Document type: Other Language: en Some participants also described how such in-group sexual colourism created tensions between Black people and was particularly harmful to Black women with darker skin shades. For example, Erika, a Millennial who said her skin was dark brown, described feeling pain and marginalisation due to experiencing colourism from men with dark skin. She said: Section title: “The only dark-skin people we like are the dark-skin men”: colourism shaping women’s experiences of romantic relationships Educational score: 1.0192204713821411 Domain: other Document type: Other Language: en Her comment that “it felt like you were not your type’s type. So, where do you kind of go from there?” conveys a concern that Black women with darker skin frequently express , namely the idea that if Black men with dark skin shun Black women of the same skin shade when it comes to dating and relationships, Black women with darker skin are overlooked in the relationship market. Erika points out that it is women with light skin, “the right kind of hair” and green or light eyes that are desired, rather than women who look like her. Section title: “The only dark-skin people we like are the dark-skin men”: colourism shaping women’s experiences of romantic relationships Educational score: 1.083950161933899 Domain: other Document type: Other Language: en In contrast, Jennifer, a Millennial who described her skin as “tanned,” said she was desired by Black young men, but their preference for women with light skin made her feel “uncomfortable.” Section title: “The only dark-skin people we like are the dark-skin men”: colourism shaping women’s experiences of romantic relationships Educational score: 0.99354088306427 Domain: other Document type: Other Language: en Rather than enjoying being privileged because of her light skin, she said she did not feel particularly privileged and found the way her skin shade and mixedness were lauded by others as special to be “ignorant.” Jennifer was not alone in considering that light skin is not always a privilege. Some of the mixed Black-White participants said they were fetizishized and excluded from belonging to an imagined community of Black people, which we discuss in detail elsewhere . Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 1.0296452045440674 Domain: other Document type: Other Language: en In this section, we explore how the inequalities that colourism engendered led to tensions between Black women with dark skin and Black and mixed Black-White women with lighter skin shades. We begin by examining perceptions that women with light skin consider themselves superior to their counterparts with darker skin. We then analyse narratives that argue that sexual colourism leads to competition between women with dark skin and those with light skin, who are privileged in the relationship market. We conclude by suggesting that tensions between women might be exacerbated due to the perception that mixed Black-White women downplay colourism. Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 1.7605350017547607 Domain: other Document type: Study Language: en Some participants described how Black and mixed Black-White women with light skin were stereotyped as being superior and argued that this harmed relations between women of different skin, a finding that resonates with Spratt’s study. She found that Black women with dark skin argued that women with light skin felt that they were “special” and might avoid engaging with Black women with dark skin, preferring the company of White women and those with light skin, whom they considered their equals. Similarly, in our study, Erika suggested that women who were Mixed and/or had light skin felt superior, which led to tensions: Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 1.28752601146698 Domain: other Document type: Other Language: en In parallel to this, women in the study with lighter skin described how they perceived they were viewed by Black women with darker skin. For example, Jennifer shared her experience as a woman with light skin who faced “resentment” from Black women with darker skin who assumed she was conceited because her skin shade made her desirable. Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 1.0068917274475098 Domain: other Document type: Other Language: en Jennifer argued that in contrast to misconceptions about her that relate to stereotypes about women with light skin (“people automatically assume that you have that confidence”), she is “very far from being up myself,” (i.e., being conceited or arrogant). Similarly, Sofia, who also had light skin, suggested that women with skin shades similar to hers are resented due to the light-skin privilege that some benefit from and perceptions that they are “stuck up” and conceited, and “not all that” (i.e., not as special as they think they are). Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 1.2888458967208862 Domain: other Document type: Other Language: en Notably, participants in our study described how these stereotypes were not benign. For example, for Sofia, stereotypes about Black women with light skin impeded her ability to have relationships with Black women with dark skin. She said: Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 1.021813988685608 Domain: other Document type: Other Language: en Sofia’s comments highlight the barriers that colourism, and the resultant mistrust between women with dark and light skin, can create. Her narratives suggest that from her perspective, her skin shade is used to stereotype her, and she is seen as a type, rather than an individual in her own right. Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 1.056740164756775 Domain: other Document type: Other Language: en Furthermore, several of the mixed Black-White women participants from different generational cohorts said that they faced hostility from Black women with darker skin due to competition in the relationship market and resentment about their light-skin privilege. For example, Ella, who was Generation Z and described her skin shade as a “yellowy colour,” said that on social media women with light skin were constructed as denigrating Black women and “stealing” Black men: Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 0.9898486137390137 Domain: other Document type: Other Language: en Ella suggested that Black women treat her “differently” because they think she feels superior due to her light skin. The narratives in the previous section about Black men, including those with dark skin, shunning Black women and desiring Mixed women and those with light skin, help to contextualise what Ella describes as online posts about women with light skin “stealing” Black men. Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 0.9579517245292664 Domain: other Document type: Other Language: en Similarly, Grace, who was Generation X and described her skin as ‘dark for someone who was Mixed’, was more explicit about the way in which she feels Black women treat her. She said: Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 0.9913680553436279 Domain: other Document type: Other Language: en Grace argued that Black women subjected her to “microaggressions” and “passive aggressiveness” and made her feel that she should not date Black men or frequent what she described as “Black spaces” due to “the competitiveness between skin shades.” In doing so, she seemed to be describing hostility driven by colourism-fuelled competition, and particularly competition for Black men, between Black and Mixed women, or those with dark skin and those with lighter skin shades. Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 0.997657060623169 Domain: other Document type: Other Language: en Jennifer, a Millennial, made similar points, arguing that she feels drawn into competition with Black young women who feel she is encroaching on their space, when she does not wish to compete. Her comments resonate with Campion’s argument that Black and mixed Black-White women “can, unwillingly, find themselves pitted against other women in competition for male approval.” Jennifer said: Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 1.0055444240570068 Domain: other Document type: Other Language: en Her description of Black young women giving her “the side eye” suggests that she faces microaggressions in a similar way to Grace. Despite stating that she does not want to be in competition with Black women, the fact that she has tanned skin and is Mixed ethnicity, means that she has an advantage in the relationship market over Black women who may consider her their competitor. Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 1.05437433719635 Domain: other Document type: Other Language: en Further complicating relationships between Black women with dark skin and Black and mixed Black-White women with light skin, some participants with darker skin shades argued that those with lighter skin often downplay colourism, and their perspectives tend to be prioritised over those of Black women who experience the negative effects of the prejudice. For example, Bilal said: Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 0.9974658489227295 Domain: other Document type: Other Language: en Bilal suggests that Black men choose to listen to women of Mixed ethnicity and those with light skin who argue that colourism claims are “exaggerated” and only in Black women’s heads, rather than listening to the perspectives of Black women with darker skin, who are lower down the hierarchy. While he does not discuss the impact of this on relations between women of different shades, from the preceding narratives it seems likely that such scenarios would cause frustration and potentially resentment. Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 0.977683424949646 Domain: other Document type: Other Language: en In a similar way, Erika said “a lot of the time it’s denied,” with people declaring that they just have a particular preference. As Silvestrini argues, sexual colourism “reproduces hierarches of race and power under the illusion of personal preferences towards racial ‘types’.” Erika described the work she does to try and get people to understand that if everyone has the same narrow preference then something more is going on. However, she suggested that people can perceive her to be “bitter” or an “angry Black woman” for bringing up the topic. Section title: “Competitiveness between skin shades”: the pernicious impact of colourism on relationships between women Educational score: 1.0029633045196533 Domain: other Document type: Other Language: en Erika suggested that women with dark skin are seen as jealous (“Dark-skin women cannot accept that light-skin women are more desirable”). In doing so, she both highlighted tensions between women of different shades, as discussed above, and underlined how difficult it is to challenge colourism among Black people when the views of those critiquing the prejudice are trivialised. Section title: Small acts of resistance: rejecting colourist ideology and embracing Black beauty Educational score: 1.001970648765564 Domain: other Document type: Other Language: en This final section focuses on participants’ narratives about countering colourism in the context of gendered appearance ideals and self-worth. Accordingly, we examine how participants discussed the need to resist colourist appearance ideals through different strategies including critiquing and rejecting societal appearance ideals, education, and taking pride in their skin shade and appearance. Section title: Small acts of resistance: rejecting colourist ideology and embracing Black beauty Educational score: 1.0091415643692017 Domain: other Document type: Other Language: en Some participants emphasised the importance of rejecting racist and colourist ideologies that tell Black people with dark skin that they are inferior and ugly. Malakai, for example, emphasised the need critically to appraise appearance ideals and to recognise the contradictions inherent in the fact that physical features that are associated with, but not privileged in, Black bodies, are increasingly celebrated in White bodies. Implicitly, Malakai called for Black people to reject the dominant racist and colourist messages. He said: Section title: Small acts of resistance: rejecting colourist ideology and embracing Black beauty Educational score: 1.033776879310608 Domain: other Document type: Other Language: en Malakai suggests that mainstream appearance ideals are racist and colourist with White women desiring (and having surgery to achieve) features that are denigrated when associated with Black women. Although he does not use the term, what he is describing is a phenomenon known as “blackfishing,” which includes “altering physical appearances through physical and digital means” to appropriate black aesthetics . When he says, “[T]hey make you feel worthless while they are stealing what you have. Look at it differently. Think about it,” he invites an imagined audience of Black women to reflect on the injustice and contradictions inherent in appearance ideals. “Look at it differently” may be a call for Black women to reevaluate both their perceptions of beauty and their self-perceptions. Section title: Small acts of resistance: rejecting colourist ideology and embracing Black beauty Educational score: 0.9737842679023743 Domain: other Document type: Other Language: en Just as Malakai called for Black women to reflect and change their perspectives, Portia, who was in an older generational cohort, described her ‘journey’ of “re-educat[ing]” and “de-brainwash[ing]” herself. Section title: Small acts of resistance: rejecting colourist ideology and embracing Black beauty Educational score: 1.0117331743240356 Domain: other Document type: Other Language: en Portia argued that colourism is still prevalent, and it is not clear that progress is being made at a collective level, though she has educated herself and worked on addressing her internalised colourism in a way that arguably enable her to “[l]ook at it differently.” As Glenn argued, attempts to address colourism are often individualised and framed around “re-education” that focuses on broadening conceptions of beauty and desirability to include dark skin so that whiteness and light skin are no longer viewed as the “dominant standard.” Section title: Small acts of resistance: rejecting colourist ideology and embracing Black beauty Educational score: 1.002158522605896 Domain: other Document type: Other Language: en Other women focused on taking pride in the darkness of their skin and the way that they looked in a moment when there is a renewed move to celebrate natural Black beauty. For Kelly, ensuring she was “presentable” was an important way to challenge negative colourist narratives about Black women with dark skin, demonstrating that “we are beautiful as well.” Her comments evoke the “Black is Beautiful” slogan from the 1960s and 1970s Black Consciousness and Black Power movements, which protested against institutional racism and sought to celebrate Black features, including dark skin, Afro hair and Black cultures . Kelly said: Section title: Small acts of resistance: rejecting colourist ideology and embracing Black beauty Educational score: 0.9922329783439636 Domain: other Document type: Other Language: en Kelly struck a defiant tone as she argued that she took pride in her appearance to emphasise the beauty in women with dark skin that mainstream colourist narratives seek to deny. Collins asserts that when Black women draw on whatever is at their disposal “to be self-defined and self-valuating [ sic ] and to encourage others to reject objectification, then Black women’s everyday behaviour itself is a form of activism.” She suggested that just the act of viewing oneself “as fully human, as subjects,” is enough to become an activist even if only in a limited way. In her narrative, Kelly rejects definitions of Black women with dark skin as ugly or unattractive and asserts her own “definition” of Black women with dark skin as beautiful, which could be seen as a form of resistance or “activism.” Section title: Small acts of resistance: rejecting colourist ideology and embracing Black beauty Educational score: 0.9835864901542664 Domain: other Document type: Other Language: en When asked how she felt about her skin shade, Destiny said, “I love it, I always have.” She said, “I was lucky growing up in somewhere like [area in London] where everyone was from a different place […]. [I]n places like [area in London], Blackness is celebrated, so I was really happy.” Her appreciation for her skin shade was reflected in her description of how she puts on makeup: Section title: Small acts of resistance: rejecting colourist ideology and embracing Black beauty Educational score: 0.9760338664054871 Domain: other Document type: Other Language: en In a similar way to Kelly, Destiny took pride in her appearance and presented herself to the world in a way that emphasised the fact that she saw darker skin as valuable. Section title: Small acts of resistance: rejecting colourist ideology and embracing Black beauty Educational score: 1.0015534162521362 Domain: other Document type: Other Language: en Ekow pointed to changes in perceptions around beauty, suggesting that there has been a shift in how Black people both see and accept themselves: Section title: Small acts of resistance: rejecting colourist ideology and embracing Black beauty Educational score: 0.9813138842582703 Domain: other Document type: Other Language: en He contrasts Black people’s comments about feeling inferior and unattractive as they were coming of age, with what he perceives to be them “beginning to feel and accept themselves” much more. His comments should be contextualised against a backdrop of digital and hybrid movements that centre on celebrating black beauty and natural hair. Such movements include the ‘natural hair’ movement and the “#BlackGirlMagic” hashtag, which “insists on the visibility of black women and girls as beauty subjects and aspirational figures in the wider culture” in an effort to challenge discourses that construct Black women as dysfunctional, unattractive and “social failures” . Section title: Discussion Educational score: 1.2158180475234985 Domain: other Document type: Study Language: en In this paper, we examined the complex processes through which colourism situates Black and mixed Black-White women in contrasting positions in beauty and desirability hierarchies based on their skin shade. We took an intersectional approach to our analysis to allow us to be particularly attentive to participants’ positionality in terms of gender, skin shade, and where possible, their generational cohort. Our findings contribute to international work on colourism by presenting a British perspective, and providing an in-depth account of colourism’s role in the heterosexual relationship market, and the effect of sexual colourism on platonic relationships between Black and mixed Black-White women. Section title: Discussion Educational score: 1.4020625352859497 Domain: other Document type: Study Language: en Women and men in our study argued that gendered colourism in the media, and society more broadly, could be internalised by Black people, leading both to negative self-perceptions among women with dark skin, and negative perceptions of women with darker skin shades. This supports US qualitative research with Black women and adolescent Black girls that emphasises the negative impact of gendered colourism on participants . Triangulating our findings with those in the US with different age groups underscores the similarity of experiences of Black girls and women across the Atlantic. Section title: Discussion Educational score: 1.575421690940857 Domain: other Document type: Study Language: en Notably, alongside a lack of representation of women with dark skin, participants in our study often highlighted the lack of representation of Afro hair in media portrayals of female beauty. This is consistent with work by Joseph-Salisbury and Connelly , who argue that Black hair practices need to be contextualised within a racialised social structure that privileges White appearance ideals and stigmatises physical features associated with Black people, such as skin colour and hair. Together, this highlights the need to consider hair texture, alongside skin shade, in analyses of how gendered colourism affects Black women. Section title: Discussion Educational score: 1.4037718772888184 Domain: other Document type: Study Language: en Women and men in our study also described how gendered colourism, perpetuated by appearance ideals in the media, is internalised and enacted by Black men, informing their (heterosexual) relationship choices. Specifically, Black men rejected Black women with dark skin in the heterosexual relationship market, preferring Black women with light skin or White women. Participants described how this led to competition between Black women with dark skin and Black and mixed Black-White women with light skin. This is consistent with Campion , who asserted that Black and mixed Black-White women can be drawn into competition for male attention. Campion argued that “the systematic racism, sexism and colourism experienced by darker skinned Black women can create frustrations which, in some cases, can result in ‘interpersonal conflict’ with Black mixed race women.” Section title: Discussion Educational score: 1.0235170125961304 Domain: other Document type: Other Language: en Some of our participants described small acts of resistance whereby Black people, including those with dark skin, rejected colourist ideology and embraced notions of Black beauty. While this was presented as a sign of hope in the face of colourist controlling images, individualised rejection of colourism is insufficient to combat the prejudice. What is required is the dismantling of the structures that sustain racism and colourism. As Glenn argues, “focusing only on individual consciousness and motives distracts attention from the very powerful economic forces that help to create the yearning for lightness and that offer to fulfil the yearning at a steep price.” Multinational corporations profit from the lucrative global skin lightening industry and are therefore invested in perpetuating colourism in order to create and sustain demand for products that depend on inequities and insecurities created by colourism . Addressing colourism will require educating and regulating both the corporations that profit from it and the institutions that help to sustain it . Section title: Limitations Educational score: 2.320546865463257 Domain: biomedical Document type: Study Language: en There are three key limitations of the study worth consideration. First, we employed convenience sampling, and our resulting sample included almost twice as many Black or mixed Black-White women with light skin as Black women with darker skin shades. This means that we have an imbalance in the sample that may have skewed the data on perceptions of relationships between women with dark and light skin and how they are affected by colourism. Relatedly, fewer men participated than women, meaning that we had fewer male perspectives to draw on. Second, our findings may be affected by a selection bias based on our recruitment strategy in which minoritised ethnic people were invited to take part in an interview study about “skin shade.” We may, therefore, have attracted those for whom skin shade is particularly salient and who would be happy to talk about the role of skin shade in their lives. Section title: Limitations Educational score: 1.449609398841858 Domain: other Document type: Study Language: en Third, it would have been valuable to include a specific question on relationships with women of different skin shades to capture more perspectives on how colourism affects interpersonal dynamics between Black and mixed Black-White women with different skin shades. However, the fact that many participants commented on this despite not being asked directly, could be viewed as a strength of this inductive analytical work. Finally, though we collected age data, the spread of participants’ ages in conjunction with our relatively small sample size meant that we were often unable to make meaningful comparisons or interpretations by age. Section title: Future directions Educational score: 1.078596591949463 Domain: other Document type: Other Language: en Colourism research is still relatively nascent in the UK. Therefore, there are several directions of research that could build upon the current work focused on colourism and romantic relationships in the UK. First, it would be valuable to conduct a larger in-depth UK study with Black and mixed Black-White women, capturing the perspectives of more women with dark skin and specifically asking women of all shades how they perceive and relate to Black and Mixed women of different shades, as well as asking them about their desire and experiences in the relationship market. Second, it would be beneficial to conduct similar research on colourism and relationships with minoritised ethnic people of other ethnicities in the UK. Third, understandings of colourism in the context of relationships would benefit from more research with minoritised ethnic people who occupy marginalised identities related to sexual orientation and gender to expand intersectional analyses. Fourth, more nuanced intersectional understandings of colourism and relationships could be gained by considering personal characteristics such as social class and generation status. Section title: Conclusion Educational score: 1.3489910364151 Domain: other Document type: Study Language: en This study highlights how colourist and gendered appearance ideals can affect Black and mixed Black-White women’s experiences in the heterosexual relationship market. We argued that Black women with dark skin and Afro hair were seen as unattractive due to sexual colourism, in contrast to the perceived beauty of mixed Black-White women, who were desired and sometimes objectified. Participants argued that dominant media constructions of white and light skin as beautiful and desirable, and dark skin as the opposite, could lead Black women with dark skin to internalise the idea that they are ugly, while Black men shunned them in favour of women with lighter skin in the romantic relationship market. Section title: Conclusion Educational score: 1.3768230676651 Domain: other Document type: Study Language: en We found that sexual colourism damaged platonic relationships between women of different shades, some of whom felt they were thrust into competition with each other, creating resentment and mistrust. In this way, the study reveals some of the subtle, less tangible effects of colourist, gendered appearance ideals, which shape Black and mixed Black-White women’s realities. Our intersectional approach to analysing how sexual colourism affects Black and mixed Black-White people, and particularly women, was valuable as it enabled us to be attentive to the complex ways in which intersecting factors such as gender, racialisation and skin shade affected both our participants’ experiences and the possibility of heterosexual and platonic relationships. In particular, it allowed us to highlight the detrimental impact of sexual colourism on Black women with dark skin. | Other | other | en | 0.999998 |
PMC11696287 | Section title: Introduction Educational score: 4.254528999328613 Domain: biomedical Document type: Review Language: en The interplay between motor experience and cognitive functions has garnered significant interest within the realms of sports science and neuroscience . Research has shown that cognitive functions, such as attention, executive control, and working memory, can benefit from sports practice and long-term sport-related training . For instance, Voss et al. provide a comprehensive review indicating a positive relationship between motor training and cognitive performance. However, a critical question remains: does expertise in specific sports disciplines enhance performance on general cognitive tasks unrelated to the sports context? Investigating this question could provide deeper insights into how sports practice influences cognitive processes beyond the sports setting. Such understanding may also offer valuable guidance for athletic programs and physical education curricula. For instance, if certain types of sports are linked to more significant cognitive benefits, educators and public health officials could advocate for these activities among adolescents, individuals with cognitive deficits, and the elderly. While studies demonstrate that elite athletes show superior proficiency in cognitive tasks involving problem-solving, motor planning, and decision-making compared to non-athletes , it remains unclear how these benefits vary across different types of sports. This observed cognitive advantage aligns with the cognitive skill transfer theory, which posits a ‘broad transfer’ mechanism whereby the intensive practice of specific skills, such as those developed through sports, enhances cognitive components utilized beyond the sports context . Furley and Memmert support this theory, noting that sports training can improve individual cognitive functions applicable in various non-sport settings. In terms of attentional functioning, studies have shown that athletes perform better in attention-demanding tasks than non-athletes, with differences in brain activation and deactivation patterns . Section title: Introduction Educational score: 3.7406630516052246 Domain: biomedical Document type: Study Language: en While it is established that athletes exhibit enhanced attentional processing , limited research has explored whether the type of sport, particularly open- versus closed-skill sports, influences the degree of attentional enhancement. The cognitive and motor demands differ significantly between sports, which may differently influence attentional processes . Sports can generally be divided into two categories: open skill and closed skill. Open-skill sports, such as basketball, tennis, and fencing, require athletes to respond to a constantly changing, unpredictable environment . Conversely, closed-skill sports like running and swimming take place in more predictable, consistent settings . Athletes in open-skill sports often develop greater flexibility in visual attention, decision-making, and action execution compared to their counterparts in closed-skill sports due to the complex cognitive demands of adapting to an ever-changing environment . Section title: Introduction Educational score: 3.903639793395996 Domain: biomedical Document type: Study Language: en Previous research suggests that open-skill sports may lead to greater enhancements in cognitive functions due to the complex cognitive demands of adapting to dynamic environments . This distinction is underscored by meta-analyses showing that athletes from open-skill sports perform better on cognitive tasks than those from closed-skill sports, highlighting the importance of comparing sport types . However, most studies have assessed these cognitive benefits using tasks that require high levels of cognitive control, such as inhibitory control , working memory , visuospatial working memory , interference control , cognitive flexibility , visuospatial attention and memory processing , and visuospatial attention . Furthermore, the majority of these studies have focused on behavioral outcomes, with relatively few investigating the underlying neurophysiological mechanisms. As a result, there is limited understanding of the neuroelectric processes that may differentiate cognitive benefits in open-skill versus closed-skill sports. Section title: Introduction Educational score: 4.361940860748291 Domain: biomedical Document type: Study Language: en While behavioral studies offer insights into the cognitive benefits observed in athletes, they do not adequately probe the neural mechanisms that support these benefits. Deriving from electroencephalogram (EEG), P3 (also known as P300) is a positive ongoing deflection of brain potential peaking between 300 and 800 ms after the stimulus onset and indicates the updating of mental representation as a result of top-down controlled attention reacting to a bottom-up stimulus . P3 has widespread and synchronous neural generators centered at the cingulate cortex linking frontal-mediated attention and updating to memory storage operated in the temporal/parietal brain areas . The neural substrates of P3 correspond with the attentional processing. P3 can be elicited during tasks requiring attentional processes (e.g., auditory oddball task), with increasing amplitude reflecting greater attentional resources allocated to processing the target stimulus and decreasing latency reflecting a faster speed of stimulus categorization . More efficient allocation of attentional resources to the stimulus, as reflected by larger P300 amplitudes, was reported for all regular exercisers compared with those who did not exercise regularly . Previous research utilizing ERPs has also indicated that prolonged training in sports demanding rapid responses to dynamic environments may foster several neurocognitive enhancements. These enhancements include additional cognitive benefits in areas such as inhibitory control and error processing, as evidenced by decreased N2 and increased P3 amplitudes ; heightened task-oriented attention, indicated by increased P3 amplitude ; and more efficient cognitive processing, reflected by shorter P3 latency . Section title: Introduction Educational score: 4.053962707519531 Domain: biomedical Document type: Study Language: en ERP studies have consistently demonstrated that open-skill sports, as compared to closed-skill sports, are associated with enhanced cognitive performance and neural efficiency, particularly in older adults. These benefits are often observed in areas such as inhibitory control, cognitive flexibility, and visuospatial attention, as evidenced by increased P3 amplitudes in ERP measurements . For example, open-skill exercisers display faster response times, improved inhibitory control, and heightened error processing in tasks like Stroop and task-switching, reflecting better cognitive flexibility and attentional control wang. Additionally, open-skill sports appear to promote more efficient resource allocation for tasks requiring high cognitive control, as seen in larger P3 amplitudes . These findings suggest that the complex and adaptive demands of open-skill sports may confer greater cognitive benefits than closed-skill sports, particularly in enhancing attentional and executive functions. Section title: Introduction Educational score: 4.07637882232666 Domain: biomedical Document type: Study Language: en The purpose of this study was to examine the relationship between motor skill type and attentional processing efficiency in young adults using behavioral and neuroelectric measures. Given that previous research suggests that sports incorporating higher attentional demands can enhance attentional functioning , we hypothesized that individuals who regularly engage in physical exercise would exhibit shorter response time, shorter P3 latency, and increased P3 amplitude compared to those who exercise irregularly (i.e., control). We also expected that participants involved in open-skill sports would demonstrate shorter response times, shorter P3 latency, and larger P3 amplitude than those engaged in closed-skill sports. The findings of this study are expected to advance our understanding of the generalizable cognitive benefits of motor skill experience and have practical implications for designing effective training and rehabilitation programs. Section title: Participants Educational score: 3.8711354732513428 Domain: biomedical Document type: Study Language: en Ninety-three participants were recruited from Assiut University and classified into three groups: open-skilled, closed-skilled, and control participants ( Table 1 ). All participants were right-handed, with normal or corrected-to-normal visual acuity, and had no history of neurological disorders, cardiovascular diseases, or clinical conditions requiring medication. None of the participants showed cognitive impairments (a score of below 27 on the Mini-Mental State Examination, MMSE) or depressive symptoms (a score of above 13 on the Beck Depression Inventory-II [BDI-II]) . The participants with expertise in open and closed skills were members of university sports teams and Assiut city sports clubs, with at least 5 years of professional training. They had won prizes in open competitions in their respective sports but had not received regular training in other sports. Among these, the open-skilled participants ( n = 31; 10 females) were from football and handball teams, and the closed-skilled participants ( n = 31; 12 females) were from swimming and track and field teams. The control group ( n = 31; 12 females) consisted of university students who had never participated in sports either professionally or at an amateur level. All three groups were age-matched. Each participant provided written informed consent. Ethical approval was received from the Institutional Review Board at the Faculty of Physical Education, Assiut University (No. AUN-PHY-JUN-ETHIC2), with all methods undertaken thereafter performed in accordance with the relevant guidelines and regulations. Section title: Electroencephalogram recordings Educational score: 4.1642255783081055 Domain: biomedical Document type: Study Language: en EEG was measured using surpass EMS biomedical, quantitative EMG/EP work station. The neuroelectric assessment was done by applying an auditory oddball task. The auditory stimuli had 10 ms r/f time and were either a 1,000 Hz (frequent) or 2,000 Hz (rare) tone (100 ms duration/80 dB SPL, 1.2–1.7 interstimulus interval), delivered binaurally through headphones and completely covering the ears . The order of stimulus presentation was randomized, with a 1-s inter-stimulus interval. Event related evoked potentials were recorded with Ag/ Ag Cl (Nihon Kohden) electrodes from standard locations using a 10–20 International system. The electrodes were placed at Fz, Cz, and Pz (active electrodes at frontal, vertex, and parietal areas), FPz (ground electrode on the forehead), and A1, A2 (reference electrode on the ear lobules). The vertical and horizontal eye movement artifacts (VEOG and HEOG, respectively) were assessed through the collection of bipolar electro-oculographic activity (EOG). The impedances were maintained below 10 kΩ. The measured variables include P3 amplitude measured in microvolt and latency measured in milliseconds. Each participant was instructed to respond to the occurrence of a rare stimulus by pressing a button with the thumb of their preferred hand, and response time and response error rates were recorded. Brain responses to rare stimuli were recorded and averaged. P3 was quantified as the most positive peak amplitude between 250 and 500 msec. A practice session was conducted to ensure comprehension of the task followed by the test session. Section title: Aerobic fitness assessment Educational score: 4.124375343322754 Domain: biomedical Document type: Study Language: en Aerobic fitness was assessed using the YMCA cycle ergometry protocol, which was recommended for submaximal exercise testing by the American College of Sports Medicine . This protocol estimates maximal oxygen consumption (VO2peak) with an electronically braked cycle ergometer (Monark Ergomedic 828E). Participants engaged in two to four 3-min cycling stages, maintaining a pedal speed of 50 rpm throughout the assessment. Initially, a workload of 0.5 kp was applied, and heart rates were monitored in the last 15–30 s of this stage. These preliminary heart rate readings were used to adjust the workloads for subsequent stages (for example, heart rates above 80 bpm led to workloads of 2.5 kp and 3.0 kp for the second and third stages, respectively; heart rates above 100 bpm resulted in workloads of 1.5 kp and 2.0 kp). The protocol concluded once participants maintained heart rates between 110 and 150 bpm during the latter stages. Additionally, both objective and subjective measures of exercise intensity were employed, utilizing a Polar heart rate monitor and the Borg 6- to 20-point Rating of Perceived Exertion (RPE) scale, respectively . VO2peak was calculated using a graphical method prediction equation that takes into account average heart rate, workload, age, and gender . Section title: Procedure Educational score: 4.108120918273926 Domain: biomedical Document type: Study Language: en In this cross-sectional study, participants were required to visit the laboratory for a single-day session, with instructions to refrain from exercising the day before their scheduled testing. Upon arrival, they provided informed consent and completed a demographics questionnaire, MMSE , Beck Depression Inventory-II (BDI-II) , as well as the Physical Activity Readiness Questionnaire . This was to identify any existing health conditions that could potentially be aggravated by the aerobic fitness assessment. Subsequently, participants were equipped with a heart rate monitor, and their height and weight were measured using a stadiometer and a digital scale (Omron Healthcare, Inc., Lake Forest, IL, USA), respectively. Following these measurements, participants were made to sit comfortably in a chair, whose back was turned toward the EEG recording machine in a semi-dark room with quiet surroundings. The participants were asked to avoid unnecessary movement and to remove all the metallic ornaments that they were wearing. After that, they performed the auditory oddball task. The participants’ aerobic fitness was assessed using a test of YMCA submaximal fitness test following the completion of the auditory oddball task to ensure that attentional functioning was not altered as a result of the fitness test . The experimental procedure is illustrated in Figure 1 . Section title: Statistical analysis Educational score: 3.211714506149292 Domain: biomedical Document type: Study Language: en All subjects’ demographic characteristics (i.e., age, years of education, amount of physical activity) and the behavioral (i.e., response time and response accuracy) and neuroelectric (i.e., P3 amplitude and latency) were described using means and SDs for each group (i.e., open-skill exercisers, closed-skill exercisers, control). Section title: Statistical analysis Educational score: 4.086709022521973 Domain: biomedical Document type: Study Language: en To confirm Pz as the region of interest to examine P3 (amplitude and latency) measures during the attentional processes in response to target stimuli, a two-way 2 (Stimulus: Target, Standard) × 3 (Electrode: Fz, Cz, Pz) repeated measure ANOVA was conducted to verify the target-specific and parietal-centered P3. Group differences in behavioral and neuroelectric measures were submitted to a one-way ANOVA method with a post-hoc Bonferroni-corrected t-test . The effect size was reported as the partial eta-square ( η 2 ). Bivariate correlations were conducted using Pearson product–moment correlations between demographic variables (age, years of education, BMI, and sex [coded as 1 = male, 2 = female]), aerobic fitness indices, and auditory oddball task performance measures. Section title: Statistical analysis Educational score: 4.090080261230469 Domain: biomedical Document type: Study Language: en Linear hierarchical regression analyses were then performed using attentional measures that were significantly correlated with aerobic fitness. Aerobic fitness measure was entered into step 2 in hierarchical regression analysis after the inclusion of demographic variables (i.e., age, education, and BMI) that were significantly correlated with attentional measures into step 1. Assumptions of linearity, equality of variance, independence, and normality were plotted, inspected, and verified using studentized residuals. A sensitivity analysis was conducted using G*Power 3.1.9.7, a widely used statistical tool for power analysis . Based on the current sample of 93 participants, with an alpha of 0.05 and power of 0.8, this analysis indicated that the present study was adequately powered to detect a small to medium effect size exceeding ƒ 2 = 0.08 for the variance explained by aerobic fitness while accounting for three demographic variables (age, education, and BMI) in the hierarchical regression analysis. All data analyses were performed in SPSS v.21 (IBM Corp., Armonk, NY, USA), utilizing a familywise alpha level of p = 0.05 ( Table 2 ). Section title: Participant demographics and aerobic fitness Educational score: 4.1121649742126465 Domain: biomedical Document type: Study Language: en Subject demographic data and their levels of physical activity and fitness are provided in Table 1 . A one-way ANOVA revealed non-significant differences in age, education, height, weight, and depression among the three groups ( p s > 0.05). As expected, the participants in the two exercise groups had significantly more exercise experience (i.e., years of regular exercise, duration per session, and frequency per week), F s (2, 90) ≥ 138.85, p < 0.01, η 2 ≥ 0.76, and greater levels of physical activity (i.e., METs), F (2, 90) = 108.39, p < 0.01, η 2 > 0.71, and higher aerobic fitness, F (2, 90) = 96.60, p < 0.01, η 2 > 0.68, than those in the control group. Participants in the control group also had greater BMI, F (2, 90) = 9.47, p < 0.01, η 2 > 0.17, than those in the exercise groups. A post hoc comparison demonstrated non-significant differences between the open- and closed-skill exercise groups in these indices ( t s ≥ 0.19, p > 0.05). Section title: Attentional function Educational score: 4.129673957824707 Domain: biomedical Document type: Study Language: en The behavioral and neuroelectric data of the three groups are presented in Table 2 . A one-way ANOVA revealed non-significant differences in response time, response accuracy, and P3 latency among the three groups ( p s > 0.05). Participants in the open-skilled and closed-skilled groups exhibited larger P3 amplitude, F s (2, 90) = 15.07, p < 0.01, η 2 ≥ 0.25, than those in the control group. A post hoc comparison demonstrated non-significant differences between the open- and closed-skill exercise groups in P3 amplitude ( t = 0.46, p > 0.05). Figure 2 shows the mean P3 amplitude in the three groups. Section title: Bivariate correlations Educational score: 4.040106773376465 Domain: biomedical Document type: Study Language: en Table 3 summarizes the results of the initial Pearson product–moment correlations among age, education, BMI, physical activity (IPAQ), aerobic fitness (YMCA), and behavioral and neuroelectric measures of attention (P3 component). The results show that aerobic fitness was associated with greater P3 amplitude ( r = 0.48). Accordingly, P3 amplitude was used as a dependent variable in the regression models. P3 amplitude was significantly correlated with age, education, and BMI ( r s ≥ 0.34). Because of these detected associations, age, education, and BMI were controlled in the regression analyses. Section title: Hierarchical regression analyses Educational score: 4.071225643157959 Domain: biomedical Document type: Study Language: en Table 4 summarizes the hierarchical regression results for P3 amplitude. Figure 3 illustrates the association between aerobic fitness and P3 amplitude. The hierarchical regression analysis indicated that young adults with higher aerobic fitness exhibited greater P3 amplitude, β = 0.39, t = 4.32, p < 0.01, Cohens’ ƒ 2 = 0.20, after controlling for age, education, and BMI . Section title: Discussion Educational score: 4.131845474243164 Domain: biomedical Document type: Study Language: en This study provides new insights into the association between motor skill experience and attentional functioning using behavioral and neuroelectric measures in healthy young adults. Specifically, we investigated whether cognitive benefits associated with sports differ based on the type of motor skills (open vs. closed) and whether these benefits are observable in non-sport-specific cognitive tasks. Our results indicate that motor skill experience, regardless of whether it involves open or closed skills, is associated with larger P3 amplitudes, suggesting enhanced attentional resource allocation. Additionally, hierarchical regression analysis confirmed a positive correlation between P3 amplitude and aerobic fitness, emphasizing the critical role of physical fitness in cognitive processing. These findings suggest that attentional enhancements related to sports can extend beyond the sports context, regardless of sport type, and highlight the importance of aerobic fitness in attentional functioning. Section title: Discussion Educational score: 4.070462226867676 Domain: biomedical Document type: Study Language: en Contrary to our hypothesis, we found no significant differences in attentional measures (i.e., response time, response accuracy, P3 amplitude) between open-skill and closed-skill sports participants. This finding is consistent with studies that have shown similar effects on visuospatial attention and processing speed between open- and closed-skill exercises . However, other research has suggested that open-skill sports, with their dynamic and unpredictable nature, confer additional cognitive benefits, such as enhanced cognitive flexibility, inhibitory control, and decision-making . These differences are attributed to the increased cognitive demands of adapting to changing environments during open-skill sports compared to the repetitive and predictable nature of closed-skill exercises . A systematic review further supports the idea that open-skill exercises are more effective in improving certain aspects of cognitive function compared to closed-skill exercises . The lack of significant differences in this study may be due to the specific attentional measures assessed, as both exercise types can enhance attention through different mechanisms—open-skill sports through adaptability to changing stimuli and closed-skill sports through focused, sustained attention. Accordingly, the specific cognitive domains being assessed might significantly influence the outcomes of studies comparing open-skilled and closed-skill exercises. Executive function tasks, which involve higher-order cognitive processes, are more likely to reveal differences between the two types of exercise due to the complex and adaptive nature of open-skilled activities. Section title: Discussion Educational score: 3.873861074447632 Domain: biomedical Document type: Study Language: en The limited evidence for superior cognitive benefits of open-skill sports in young adults in the current study could also be attributed to the fact that cognitive functions typically peak during young adulthood, reducing the potential for further enhancements through sports training . Therefore, as such, the differences in cognitive benefits between open- and closed-skill sports may be more pronounced in populations with developing or declining cognitive abilities. Future studies should include long-term interventions across different age groups to better assess the potential differential effects of open- and closed-skill sports . Section title: Discussion Educational score: 4.103301525115967 Domain: biomedical Document type: Study Language: en Our study further demonstrated that both sports groups had larger P3 amplitudes compared to the control group, replicating findings from previous research linking sports participation to enhanced P3 amplitudes . The positive relationship between aerobic fitness and P3 amplitude observed in our regression analysis aligns with existing literature on the neurocognitive benefits of aerobic fitness . This is in line with a growing body of studies that indicate that increased P3 amplitude after moderate to vigorous aerobic exercise . The P3 component is linked to attentional processing and working memory, with larger P3 amplitudes indicating better attentional resource allocation and more efficient cognitive processing . Thus, higher levels of aerobic fitness may contribute to improved neural efficiency even in young adults, potentially due to physiological mechanisms such as increased cerebral blood flow , the proliferation of neurotrophic factors like BDNF , and enhanced synaptic plasticity . Section title: Discussion Educational score: 4.075071811676025 Domain: biomedical Document type: Study Language: en A key strength of our study is its contribution to the generalizability of the association between aerobic fitness, motor skill experience, and P3 amplitude across diverse populations. While existing evidence on the positive relationship between aerobic fitness, sports participation, and P3 has primarily focused on Western or Asian populations , our findings among Egyptian participants, who are Arabic native speakers, extend this association to a Middle Eastern context. This cross-cultural evidence supports the broader applicability of the cognitive benefits of aerobic fitness and sports participation, adding valuable insights into how these associations hold across different linguistic and cultural backgrounds. Section title: Discussion Educational score: 4.0865607261657715 Domain: biomedical Document type: Study Language: en Despite the valuable insights gained from this study, several limitations must be acknowledged. First, the cross-sectional design limits our ability to infer causality regarding the relationship between motor skill type and attentional processing. Longitudinal intervention studies are needed to establish causality and to understand the long-term effects of different types of sports engagements on cognitive functions. Second, although the auditory oddball task is a well-established measure of attentional processing, it may not fully capture the complexity of attentional engagement required in real-world scenarios. Future studies should incorporate assessments of different types of attention (e.g., divided attention, sustained attention) using neuroelectric measures to provide a more comprehensive evaluation of attentional functioning. Additionally, while presenting grand average waveforms for the P3 ERP component would enhance the visual representation of our data, this was not feasible with our EEG system, which provides only individual participant waveforms and lacks data extraction capabilities for external analysis. Future studies should consider EEG systems that support data extraction to enable group-averaged waveform presentations. Lastly, although we controlled for several confounding factors (age, education, and BMI), other variables that may influence cognitive performance, such as dietary habits , sleep quality , and genetic predispositions , were not accounted for in this study. Addressing these limitations in future research will help to further elucidate the complex relationship between physical exercise and cognitive health. Section title: Conclusion Educational score: 4.167576789855957 Domain: biomedical Document type: Study Language: en This study advances our understanding of the relationship between motor skill experience, aerobic fitness, and attentional processing by examining these factors in a sample of young adults from an Egyptian population. Our findings demonstrate that both open- and closed-skill sports are associated with enhanced attentional engagement, as reflected by increased P3 amplitudes, suggesting that the attentional benefits derived from motor skill experience extend beyond the sports context itself. Additionally, the positive association between aerobic fitness and P3 amplitude underscores the role of physical fitness in supporting cognitive efficiency, highlighting aerobic fitness as a potential contributor to attentional enhancement. While no significant differences in attentional measures were observed between open-skill and closed-skill athletes, this study emphasizes the need for further research into how different types of sports may affect specific cognitive domains, particularly those requiring complex, adaptive attentional processing. Our findings, including participants from a Middle Eastern context, contribute to the generalizability of prior research largely conducted in Western and Asian populations, supporting a broader applicability of the cognitive benefits of motor skills and fitness. Future longitudinal studies should explore these relationships across diverse age groups and real-world cognitive tasks to deepen our understanding of how sports engagement and fitness shape cognitive health over time. | Review | biomedical | en | 0.999995 |
PMC11696350 | Section title: Description Educational score: 4.17887544631958 Domain: biomedical Document type: Study Language: en We propose a gene model for the D. yakuba ortholog of the D. melanogaster Ribosomal protein S6 kinase ( S6k ) gene. The genomic region of the ortholog corresponds to the uncharacterized protein LOC6533108 in the Dyak_CAF1 Genome Assembly of D. yakuba . This model is based on RNA-Seq data from D. yakuba and S6k in D. melanogaster using FlyBase release FB2022_04 . Section title: Description Educational score: 3.5126209259033203 Domain: biomedical Document type: Other Language: en Ribosomal protein S6 kinase is part of the insulin signaling pathway downstream of the target of rapamycin ( dTOR ) , homologous to mammalian p70S6k . S6k is a serine/threonine kinase in Drosophila melanogaster and acts as a regulator of cell size , as well as innate immunity and senescence . The species summary can be found in the box above. Section title: Description Educational score: 1.4392244815826416 Domain: biomedical Document type: Other Language: cy Synteny Section title: Description Educational score: 4.555229663848877 Domain: biomedical Document type: Study Language: en The target gene, S6k , occurs on chromosome 3L in D. melanogaster and the gene CG42272 nests within it. S6k is flanked upstream by mad2 ( mad2 ) and krishah ( kri ) and downstream by Tektin C ( Tektin-C ) and Bre1 ( Bre1 ). The tblastn search of D. melanogaster S6k-PA (query) against the D. yakuba Genome Assembly (database) placed the putative ortholog of S6k within scaffold chromosome 3L at locus LOC6533108 — with an E-value of 3e-69 and a percent identity of 43.04%. Furthermore, the putative ortholog of CG42272 , nests within LOC6533108 and is flanked upstream by LOC6533112 and LOC6533111 , which correspond to mad2 and kri in D. melanogaster (E-value: 6e-153 and 0.0; identity: 96.62% and 98.08%, respectively, as determined by blastp ). The putative ortholog of S6k is flanked downstream by LOC6533107 and LOC6533106 , which correspond to Tektin-C and Bre1 in D. melanogaster (E-value: 0.0 and 0.0; identity: 100.00% and 98.28%, respectively, as determined by blastp ). The putative ortholog assignment for S6k in D. yakuba is supported by the following evidence: The genes surrounding the S6k ortholog are orthologous to the genes at the same locus in D. melanogaster and local synteny is completely conserved, supported by results generated from blastp , so we conclude that LOC6533108 is the correct ortholog of S6k in D. yakuba . Section title: Description Educational score: 1.2832227945327759 Domain: biomedical Document type: Other Language: de Protein Model Section title: Description Educational score: 4.21395206451416 Domain: biomedical Document type: Study Language: en S6k in D. yakuba has two mRNA isoforms ( S6k-RA; S6k-RB ) that encode one unique protein-coding isoform . The gene contains ten CDSs. Relative to the ortholog in D. melanogaster , the RNA CDS number and protein isoform count are conserved. The sequence of S6k-PA in D. yakuba has 99.19% identity (E-value: 0.0) with the protein-coding isoform S6k-PA in D. melanogaster , as determined by blastp . Coordinates of this curated gene model of S6k-PA and S6k-PB are stored by NCBI at GenBank/BankIt . These data are also archived in the CaltechDATA repository (see “Extended Data” section below). Section title: Methods Educational score: 4.43499231338501 Domain: biomedical Document type: Study Language: en Detailed methods including algorithms, database versions, and citations for the complete annotation process can be found in Rele et al. . Briefly, students use the GEP instance of the UCSC Genome Browser v.435 to examine the genomic neighborhood of their reference IIS gene in the D. melanogaster genome assembly . Students then retrieve the protein sequence for the D. melanogaster target gene for a given isoform and run it using tblastn against their target Drosophila species genome assembly on the NCBI BLAST server to identify potential orthologs. To validate the potential ortholog, students compare the local genomic neighborhood of their potential ortholog with the genomic neighborhood of their reference gene in D. melanogaster . This local synteny analysis includes at minimum the two upstream and downstream genes relative to their putative ortholog. They also explore other sets of genomic evidence using multiple alignment tracks in the Genome Browser, including BLAT alignments of RefSeq Genes, Spaln alignment of D. melanogaster proteins, multiple gene prediction tracks (e.g., GeMoMa, Geneid, Augustus), and modENCODE RNA-Seq from the target species. Detailed explanation of how these lines of genomic evidenced are leveraged by students in gene model development are described in Rele et al. . Genomic structure information (e.g., CDSs, intron-exon number and boundaries, number of isoforms) for the D. melanogaster reference gene is retrieved through the Gene Record Finder . Approximate splice sites within the target gene are determined using tblastn using the CDSs from the D. melanogaste r reference gene. Coordinates of CDSs are then refined by examining aligned modENCODE RNA-Seq data, and by applying paradigms of molecular biology such as identifying canonical splice site sequences and ensuring the maintenance of an open reading frame across hypothesized splice sites. Students then confirm the biological validity of their target gene model using the Gene Model Checker , which compares the structure and translated sequence from their hypothesized target gene model against the D. melanogaster reference gene model. At least two independent models for this gene were generated by students under mentorship of their faculty course instructors. These models were then reconciled by a third independent researcher mentored by the project leaders to produce the final model presented here. Note: comparison of 5' and 3' UTR sequence information is not included in this GEP CURE protocol. | Study | biomedical | en | 0.999997 |
PMC11696351 | Section title: Description Educational score: 4.370082855224609 Domain: biomedical Document type: Review Language: en Macrophages that destroy foreign substances by engulfing them were discovered in 1882 by Élie Metchnikoff in starfish larvae. He described the process as phagocytosis . Subsequent investigations have demonstrated that macrophages are conserved throughout metazoans, exhibiting additional functions in regulating development, tissue repair, homeostasis, and innate immunity . In triploblastic animals, phagocytic cells travel through the coelomic cavity because of an open circulatory system and remove cellular debris or pathogens . In mammals, resident tissue macrophages develop from the yolk sac and erythro-myeloid precursors during an early embryonic stage and have self-renewal capacity throughout life. Monocyte-derived macrophages are also associated with rapidly replenishing tissues, such as in the intestine . During the evolution from single-celled organisms to highly complex vertebrates, the roles of macrophages and the phagocytic process have remained largely conserved . However, the mechanism underlying phagocytic macrophage differentiation remains unclear. Section title: Description Educational score: 4.307554721832275 Domain: biomedical Document type: Study Language: en Drosophila melanogaster provides an excellent genetic model for studying macrophage development and innate immunity. It has only myeloid-type cells comprising 95% plasmatocytes, with functional similarity to mammalian macrophages; 5% crystal cells act similar to mammalian platelets; and lamellocytes are large flat cells seen only after infection or injury. As in mammals, hematopoiesis in Drosophila melanogaster occurs in multiple waves. The first wave starts from the embryonic head mesoderm and produces macrophages and crystal cells that persist into the larval and adult stages. The second wave begins at the late embryonic stage in the hematopoietic tissue, called the lymph gland. The lymph gland matures by the late third instar larval stage, and it disintegrates during metamorphosis, contributing to adult macrophages . Section title: Description Educational score: 4.18052339553833 Domain: biomedical Document type: Study Language: en Caspases are generally involved in programmed cell death. However, studies have suggested that they also play non-lethal roles in cellular differentiation and development . Recently, we reported that caspase-mediated activation of caspase-activated DNase (CAD) causes DNA strand breaks in the differentiating progenitors of the lymph gland, which are required for macrophage differentiation . Here, we extend this finding and show that caspase-mediated DNA damage is necessary for lymph gland development in other Drosophila species phylogenetically close to D. melanogaster , viz., D. ananassae , D . malerkotliana, D. bipectinata , and D. biarmipes . Section title: Description Educational score: 4.197237968444824 Domain: biomedical Document type: Study Language: en To check the effector caspase activity, we performed multiple sequence alignments of Death related ICE-like caspase (Drice) and Death caspase-1 (Dcp-1) protein sequences in these species, except for D . malerkotliana, whose genome sequence was not available in the NCBI database. We detected highly conserved sequences with more than 80% identity in the two effector caspases, Drice and Dcp-1 in the C-terminal region, which included the cleavage site tripeptide ETD region (black box, underlined), alternative cleavage site DRLD (underlined), and active site pentapeptide QACQG (black box) . Section title: Description Educational score: 4.030191421508789 Domain: biomedical Document type: Study Language: en To examine the presence of lymph glands in these species, we dissected the late third instar larvae (just before they became white pupae). As in D. melanogaster, all the four species had lymph glands at the anterior region of the dorsal side, although their sizes differed. D. ananassae, D . malerkotliana , and D. bipectinata had smaller lymph glands, whereas D. biarmipes had the same size as D. melanogaster . It is the first report demonstrating the presence of lymph glands in these Drosophila species. Section title: Description Educational score: 4.358522891998291 Domain: biomedical Document type: Study Language: en The effector caspases Dcp-1 and Drice are activated after cleavage by the initiator caspase Dronc, which generates the active large subunit that can be detected by an anti-Dcp-1 antibody , which binds only to the cleaved large subunit of both effector caspases . Therefore, to check for caspase-mediated DNA damage during lymph gland development, we co-immunostained the late third instar larval lymph glands of the four species with anti-Dcp-1 and anti-γH2Av (a marker of DNA damage response). Remarkably, the lymph glands of all the species studied exhibited effector caspase activity and DNA damage . High-magnification images confirmed that caspase activity (Dcp-1 staining) and DNA damage (γH2Av staining) were present in the same cells . However, the number of Dcp-1 and γH2Av positive cells in the three species having smaller lymph glands (measured by DAPI volume) was lesser than in D . melanogaster . A comparison of the ratio of Dcp-1-positive cells with the DAPI volume of the lymph gland revealed that caspase activity was higher in D. ananassae , equal in D. malerkotliana , and low in D. bipectinata and D. biarmipes . The ratio of γH2Av-positive cells with DAPI volume in D . ananassae , D . malerkotliana, and D . bipectinata was higher than that in D . melanogaster but lower in D. biarmipes . These results suggest that caspase activation and DNA damage response in the lymph glands were highly conserved among the species studied. Section title: Description Educational score: 4.294585227966309 Domain: biomedical Document type: Study Language: en Caspase-mediated programmed cell death (apoptosis) has evolved as an evolutionarily conserved mechanism to eliminate unwanted cells. However, caspases also play various non-apoptotic roles in development, cellular differentiation, and survival in invertebrates and vertebrates , including monocyte-to-macrophage differentiation in mammals . In Drosophila melanogaster , caspase-activated DNase causes DNA strand breaks that regulate macrophage differentiation . This study showed that the cellular mechanism regulating macrophage differentiation is evolutionarily conserved in Drosophila . Studies have revealed that DNA strand breaks cause changes in the chromatin landscape, triggering heterogeneous gene activation . A detailed study is required to determine how DNA strand breaks regulate the chromatin landscape and gene expression during the development of evolutionarily conserved phagocytic macrophages. Section title: Methods Educational score: 1.4046398401260376 Domain: biomedical Document type: Other Language: en Fly stocks used in the study Section title: Methods Educational score: 2.5979602336883545 Domain: biomedical Document type: Study Language: en We used Drosophila melanogaster, Drosophila ananassae, Drosophila bipectinata, Drosophila malerkotliana, and Drosophila biarmipes , all of which were available in our departmental stock collection . All flies were raised on standard fly food in an incubator (PHCBI Model #MIR-554-PE) maintained at 25°C. Section title: Methods Educational score: 2.3075449466705322 Domain: biomedical Document type: Other Language: en Drosophila larval lymph gland dissection and immunohistochemistry Section title: Methods Educational score: 4.200492858886719 Domain: biomedical Document type: Study Language: en Lymph glands of the wandering third instar larvae were dissected in chilled 1X PBS (phosphate-buffered saline) and fixed in 4% paraformaldehyde for 30 min. The tissues were then washed thrice with 0.3% PBST (0.3% Triton X-100 in 1X PBS) for 15 min each. After incubation in blocking solution (0.1% Triton X-100, 0.1% BSA, 10% FCS, 0.1% sodium deoxycholate, and 0.02% Thiomersal, in 1XPBS) for 30 min, they were incubated overnight in the desired primary antibody at 4°C, following which the tissues were washed three times with 0.3% PBST and incubated in a blocking solution for 30 min. Finally, the tissues were incubated with a secondary antibody at room temperature for 2 h, washed in 0.3% PBST three times, counterstained with DAPI , and mounted in DABCO . Section title: Methods Educational score: 2.921806812286377 Domain: biomedical Document type: Other Language: en Primary antibodies used for immunohistochemistry were rabbit anti-Dcp1 and mouse anti-γH2Av . The secondary antibodies (1:200 dilutions) were donkey anti-rabbit Alexa Fluor 555 (Invitrogen), goat anti-rabbit Alexa Fluor 647 (Invitrogen), goat anti-mouse Alexa Fluor 488 (Jackson ImmunoResearch), and goat anti-mouse Cy3 (Jackson ImmunoResearch). Section title: Methods Educational score: 1.2687739133834839 Domain: biomedical Document type: Other Language: en Microscopy, image processing, and analysis Section title: Methods Educational score: 4.050017356872559 Domain: biomedical Document type: Study Language: en Images were acquired on a Zeiss LSM-900 confocal microscope using Zen software (version 3.4) with a 40X objective and identical imaging settings for all tissues. ImageJ software (NIH, USA) was used for image processing. Adobe Photoshop software was used to assemble the figure panel. The maximum intensity projection of the middle third stack of the lymph gland was used to better represent the inside of the lymph gland. For clarity, the white dotted line in Figure 1 demarcates lymph gland boundaries. At least three independent biological replicates and three experimental replicates were analyzed. The Interactive Tree of Life (iTOL) version 7.0 (https://itol.embl.de) was used to construct the phylogenetic tree, and NCBI BLAST was used to align the sequences. Section title: Methods Educational score: 2.500972270965576 Domain: biomedical Document type: Study Language: en Quantification of lymph gland phenotypes Section title: Methods Educational score: 3.998286485671997 Domain: biomedical Document type: Study Language: en All quantifications were performed using ImageJ software (NIH, USA). The number of γH2Av and Dcp-1 positive cells was counted manually in both lobes of the primary lymph gland and analyzed separately. The procedure to determine the threshold was performed during image processing to select pixels of interest based on the intensity of the pixel values, following which the ‘‘Measure stack'' plugin was used to find each optical section's fluorescent area in DAPI staining. The fluorescent area in each optical section was added and multiplied by the stack interval (2 μm) to determine the volume . Quantification graphs were prepared using GraphPad Prism 9 software. | Review | biomedical | en | 0.999999 |
PMC11696369 | Section title: INTRODUCTION Educational score: 3.834808588027954 Domain: biomedical Document type: Other Language: en High-risk pregnancy is a condition that requires special attention in maternal and child health services, due to the high potential for complications that can affect the health of the mother and fetus. Various factors such as health history, socio-economic conditions, and limited access to comprehensive health services often worsen the condition of high-risk pregnant women 1 , 2 . To meet this challenge, the continuity of care (COC) approach is crucial in ensuring continuity of monitoring and effective interventions. COC is a concept that emphasizes the importance of continuity in the delivery of health services from pregnancy to postpartum, with the aim of increasing family independence in preventing pregnancy complications 3 , 4 . Section title: INTRODUCTION Educational score: 3.5103681087493896 Domain: biomedical Document type: Other Language: en One of the main problems in the healthcare of high-risk pregnant women is the lack of continuity of care received by women during pregnancy and postpartum. Studies show that this lack of continuity of care is often due to differences in health providers, limited access to health facilities, and lack of knowledge among mothers and families about measures that can be taken to prevent pregnancy complications 5 , 6 . This condition is exacerbated by the limited role of families in supporting pregnant women undergoing high-risk pregnancies. In fact, the role of the family is very important in ensuring that pregnant women get proper care and comply with medical advice 7 , 8 . Section title: INTRODUCTION Educational score: 3.9530038833618164 Domain: biomedical Document type: Study Language: en Recent studies have shown that implementing COC in high-risk pregnant women can lead to better pregnancy outcomes, including reduced rates of complications such as preeclampsia, preterm birth, and other labor complications 9 , 10 . COC involves multidisciplinary collaboration between midwives, doctors, and other health professionals who work together to provide coordinated and continuous care. A study by Hardiningsih et al. 11 found that pregnant women who received services using the COC approach showed increased independence in making health-related decisions, including in terms of nutritional intake, physical activity, and appropriate use of health services. In addition, this approach also helps in early detection of complications, so that medical interventions can be carried out more quickly and effectively. Section title: INTRODUCTION Educational score: 4.020567893981934 Domain: biomedical Document type: Study Language: en While the benefits of COC have been recognized in various studies, there are several challenges that require further attention. One of the main challenges is the suboptimal implementation of COC in high-risk pregnant women, especially in developing countries. Limited human resources and health infrastructure are often the main barriers to equal access to COC services, especially in remote areas 6 , 12 . In addition, most studies on COC emphasize clinical outcomes, while other important aspects such as family self-reliance in preventing complications during pregnancy are less explored. Family empowerment through education and support is a crucial component in the successful implementation of COC 13 , 14 . Therefore, further research is needed to identify the role of COC in improving family self-reliance, particularly in resource-limited countries, to ensure the effectiveness of preventing pregnancy complications in high-risk pregnant women. This study aims to evaluate the role of COC in improving family self-reliance in supporting the health of high-risk pregnant women. Section title: Research design and setting Educational score: 3.9885337352752686 Domain: biomedical Document type: Study Language: en This study used a quantitative approach with a quasi-experimental design. The design applied was a non-randomized pretest-posttest without control group design, which allows measuring changes in family self-reliance in the prevention of pregnancy complications in high-risk pregnant women before and after the continuity of care (COC) intervention without comparing with a control group. Section title: Research design and setting Educational score: 2.099112033843994 Domain: biomedical Document type: Study Language: en The study was conducted at Puskesmas Wonoayu, Sidoarjo, Indonesia, which is a primary healthcare facility serving the community in its working area. This health center was chosen because it had a sufficient number of high-risk pregnant women to meet the study inclusion and exclusion criteria. The participant recruitment process took place from February to May 2024, and data collection was conducted in stages during this period, with a one-month post-intervention follow-up to measure changes in family independence. Section title: Research design and setting Educational score: 2.3045380115509033 Domain: biomedical Document type: Study Language: en Participants in this study were pregnant women with high-risk criteria who met the inclusion requirements, such as maternal age, relevant medical history, and multiple pregnancies. Section title: Participants Educational score: 3.78487229347229 Domain: biomedical Document type: Study Language: en This study involved 134 high-risk pregnant women from the working area of Puskesmas Wonoayu, Sidoarjo, Indonesia. The sampling method used was purposive sampling, to ensure that the participants involved matched the required high-risk characteristics. Participants were divided by trimester of pregnancy to ensure representative distribution. Inclusion criteria were: pregnant women with first to third trimester gestational age; having a high-risk pregnancy (having hypertension, gestational diabetes); previous history of pregnancy complications; and being able to communicate in Indonesian. Additional criteria included willingness to involve family members in the COC intervention, as the family plays an important role in the complication prevention strategies implemented. Section title: Ethics Educational score: 1.467830777168274 Domain: biomedical Document type: Other Language: en This study was approved by the Health Research Ethics Committee of Poltekkes Kemenkes Surabaya with registration number EA/2689/KEPK-Poltekkes_Sby/V/2024 dated 13 November 2023. Before the study began, all respondents provided informed consent, after explanation of the purpose of the study, intervention procedures, and their rights as participants. Respondents had the right to withdraw from the study at any time without any consequences. The data collected from the respondents will be kept confidential and only used for research purposes. Section title: Study variables Educational score: 3.4541900157928467 Domain: biomedical Document type: Study Language: en The independent variable in this study was the COC intervention, which involves a series of ongoing health monitoring services for high-risk pregnant women. The dependent variable was the level of family role in preventing pregnancy complications, which was measured through the family’s ability to recognize risk signs, take preventive action, and proactively accompany pregnant women across the following domains: fulfilment of physiological and psychological needs; labor preparation; postpartum preparation; and preparation after the baby is born. Section title: Study variables Educational score: 3.851393938064575 Domain: biomedical Document type: Study Language: en This level of independence was assessed before and after the intervention to observe changes as a result of COC. Other factors assessed included maternal age, gestational age, socioeconomic status, medical history, and family support. Maternal age and gestational age may influence readiness to receive information and take preventive measures, while socioeconomic status relates to access to health resources and family financial preparedness. Health history, which includes the presence of chronic diseases or previous pregnancy complications, was also considered as it may increase the need for more intensive health support. Unequal family support in accompanying pregnant women was also an important variable, as family independence is highly dependent on the involvement of family members. Section title: Intervention Educational score: 3.655123233795166 Domain: biomedical Document type: Study Language: en The intervention in this study was a COC program designed to improve family self-reliance in the prevention of pregnancy complications in high-risk pregnant women. The intervention focuses on providing health education, regular monitoring, and active family involvement in the care of pregnant women during pregnancy, to reduce potential complications that may arise. The intervention was delivered in the form of face-to-face meetings and counselling sessions, where pregnant women and family members were provided with information on pregnancy danger signs, prevention measures, and the importance of ongoing health care. Section title: Intervention Educational score: 2.654813528060913 Domain: biomedical Document type: Study Language: en Participants in the COC intervention received routine pregnancy monitoring at Puskesmas Wonoayu, including health checks by midwives and other health workers. They were also provided with educational materials on danger signs during pregnancy, ways to prevent complications, and the importance of family support in maintaining the health of pregnant women. Each participant received counselling sessions conducted by an experienced midwife, who was also responsible for providing training on family health monitoring. During the intervention, families were taught how to monitor the condition of pregnant women, such as blood pressure and watch for signs of complications that should be reported immediately. Section title: Intervention Educational score: 4.093182563781738 Domain: biomedical Document type: Study Language: en To avoid any bias in the delivery of the intervention, all participants receiving COC were given similar treatment, with no other treatments or exposures that could affect the results. All participants in the intervention group followed the same program, with interventions delivered by a trained and standardized team to ensure quality delivery of information and monitoring of maternal health. To ensure consistent intervention delivery and reduce inter-operator variability, training and standardization of procedures were conducted for all health workers involved, especially the midwives who conducted the education and monitoring sessions. There were five midwives involved in data collection, and they were trained to use uniform instruments and follow the same protocol during data collection and intervention delivery. With an uncontrolled pretest-posttest design, the intervention was closely monitored to ensure that changes in family self-reliance could be attributed to COC delivery and not influenced by other uncontrolled variables. Section title: Data sources and measurements Educational score: 3.1295230388641357 Domain: biomedical Document type: Study Language: en Data sources in this study were obtained through primary data collection from high-risk pregnant women who participated in the COC program. Data were collected using a structured questionnaire completed by the participant’s family before and after the COC intervention. Data measurement was conducted using a structured questionnaire designed to assess the level of family self-reliance in preventing pregnancy complications, as well as to collect information related to the demographics, clinical conditions, and maternity history of pregnant women. Data were collected through direct interviews and questionnaires completed by participants (both pregnant women and family members involved in mentoring). Section title: Data sources and measurements Educational score: 2.49780011177063 Domain: biomedical Document type: Study Language: en The questionnaire used consisted of two main sections: the first section collected demographic data (e.g. age, education level, employment status, and socioeconomic status), while the second section measured the level of family self-reliance in supporting pregnant women. Family self-reliance was measured through a series of closed-ended questions, using a Likert scale (1–5) to assess the extent to which the family could recognize danger signs, take preventive actions, and provide support to pregnant women. Section title: Data sources and measurements Educational score: 3.7223896980285645 Domain: biomedical Document type: Study Language: en To measure the level of family self-reliance, this questionnaire was developed by the research team by considering relevant literature and previous research results. The questionnaire was pretested on a small group of pregnant women in another health center before being used in the main study to ensure clarity of questions and to identify features that required modification. Preliminary testing yielded a Cronbach’s alpha coefficient of 0.85, indicating a good level of reliability for use in this population. Validity testing was also conducted by involving experts in the field of maternal and family health to ensure the content of the questionnaire was relevant to the research objectives. The questionnaire also included open-ended questions regarding challenges faced by the family in supporting the pregnant woman and preventive measures taken. Section title: Data sources and measurements Educational score: 1.9879322052001953 Domain: biomedical Document type: Study Language: en All data were collected at two points in time: before and after the COC intervention, to assess changes in family self-reliance. The data were collected by five trained midwives, who were trained in interview techniques and questionnaire completion to ensure consistency in data collection and minimize inter-operator bias. Section title: Statistical analysis Educational score: 3.2970376014709473 Domain: biomedical Document type: Study Language: en The data obtained were analyzed using descriptive statistical tests to see the frequency distribution and sample characteristics. A comparative test using independent t-test was conducted to test the difference between the control and intervention groups. To test the difference between pretest and posttest in the intervention group, paired t-test was used. The statistical package XXX was used to perform the statistical analysis. Section title: RESULTS Educational score: 3.8700833320617676 Domain: biomedical Document type: Study Language: en Table 1 provides an overview of the characteristics of the participants of this study, which consisted of 134 pregnant women categorized as high risk. The majority of respondents were in the first trimester of pregnancy (58.2%), followed by the second (29.0%) and third (11.9%) trimesters. In terms of age, most participants (73.9%) were aged 20–35 years, which is the ideal age range for pregnancy, while only 11.19% were aged <20 years, and 14.93% were aged >35 years. The time between marriage and first pregnancy for 90% of the participants was <4 years, indicating their readiness to start a family. Pregnancy spacing showed that 47.01% of the participants had a pregnancy spacing of ≤2 years, and 97% had <4 children, indicating that many of them were relatively new expectant mothers. Lastly, 73.9% of participants had a normal pregnancy history, indicating previous positive experiences in pregnancy. Section title: RESULTS Educational score: 4.028127670288086 Domain: biomedical Document type: Study Language: en Table 2 shows significant results regarding the effectiveness of COC-based interventions on family independence in preventing pregnancy complications. Before the intervention, the mean score for physiological and psychological needs fulfilment was 17.45, which increased to 36.42 after the intervention (p<0.001). In addition, labor preparation also showed a significant increase from 11.40 to 24.38, as well as postpartum preparation from 13.00 to 28.79, and preparation after the baby is born from 13.25 to 28.75 (all p<0.001). Section title: DISCUSSION Educational score: 4.020920753479004 Domain: biomedical Document type: Study Language: en This study aimed to assess the role of COC in high-risk pregnant women in increasing family independence in preventing pregnancy complications. Based on the results obtained, this study shows that the COC intervention has a significant effect in increasing family knowledge and skills in supporting the health of pregnant women, especially in preventing pregnancy complications. Family independence in recognizing danger signs, taking preventive actions, and supporting the care process of pregnant women, increased significantly after the COC intervention. This reflects the importance of family involvement in the health management of pregnant women, especially for those in high-risk groups. Section title: DISCUSSION Educational score: 2.916908025741577 Domain: biomedical Document type: Study Language: en The success of the COC program in this study highlights the importance of integrated and sustainable health service provision, which involves not only pregnant women, but also families as part of the support system. This study shows that providing interventions that actively involve families can be an effective strategy in reducing the risk of pregnancy complications and increasing family understanding of the importance of early prevention. Section title: DISCUSSION Educational score: 3.5745022296905518 Domain: biomedical Document type: Study Language: en Based on the results of this study, it was found that the implementation of COC in high-risk pregnant women can increase the understanding and independence of families in facing various pregnancy challenges. The COC approach allows mothers and their families to receive clear and targeted information about the pregnancy risks they face, so that they can take appropriate preventive measures 15 . In this case, COC acts not only as an intervention but also as an educational tool that can increase family independence in maintaining the health of pregnant women and preventing complications. Section title: DISCUSSION Educational score: 3.647214651107788 Domain: biomedical Document type: Study Language: en Previous research has indicated that pregnant women who received COC were more confident in managing their pregnancy, especially in making decisions related to a healthy lifestyle, such as diet, physical activity, and adherence to medical advice 13 . A study by Bradford et al. 6 showed that COC is associated with improved maternal health outcomes, including reduced rates of preterm birth and other pregnancy complications. Section title: DISCUSSION Educational score: 3.941559076309204 Domain: biomedical Document type: Study Language: en The findings in this study are also supported by other studies that emphasize the importance of COC in maintaining the health of high-risk pregnant women. For example, a study found that mothers who received COC, tended to be better able to cope with health risks compared to mothers who received fragmented care 16 . In addition, other studies have also showed that COC plays a role in increasing pregnant women’s satisfaction with the health services they receive 17 . This satisfaction arises due to the close relationship between the mother and the healthcare provider, which can create a sense of security and comfort for the mother in undergoing a high-risk pregnancy. This relationship also contributes to increased family independence, as the family can be directly involved in the decision-making process based on accurate information from the provider. Section title: DISCUSSION Educational score: 2.4883530139923096 Domain: biomedical Document type: Study Language: en However, despite the overwhelming evidence showing the benefits of COC, several studies have found that COC implementation still faces challenges. Bradford et al. 6 noted that in some developing countries, limited access to sustainable health services is often a major obstacle to COC implementation, especially in remote areas. This can lead to a break in the chain of care, which in turn negatively affects the health of pregnant women 6 . Section title: DISCUSSION Educational score: 4.013439178466797 Domain: biomedical Document type: Study Language: en Improved family self-reliance through COC in this study was seen in various aspects. Families are not only more informed about their pregnancy condition, but are also more proactive in ensuring that pregnant women receive appropriate care 14 . This shows that education provided through COC can empower families to prevent complications that may arise during pregnancy, such as preeclampsia and preterm labour 13 , 14 . This family independence is in line with the concept of empowerment emphasized in maternal health literature which has noted that family empowerment through continuing education can increase family involvement in the care of pregnant women, leading to improved overall pregnancy outcomes. With a better understanding, the family can support the mother in leading a healthy lifestyle, adhering to the pregnancy control schedule, and taking prompt action if signs of complications arise 14 . Section title: Limitations Educational score: 4.073940277099609 Domain: biomedical Document type: Study Language: en Although this study shows significant benefits of implementing COC, there are some limitations that should be noted. Firstly, this study only included samples from urban areas, so the results may not be representative of high-risk pregnant women in rural or remote areas, where limited health infrastructure is often a major obstacle to optimal COC implementation. The limited number of health workers and medical facilities in these areas may affect the quality and continuity of care 18 . Secondly, the long-term impact of COC on maternal and infant health has not been fully explored in this study. Further studies are needed to evaluate how the implementation of COC may affect the health of high-risk mothers postpartum, including support for physical and mental recovery, which was also recognized as important by the study 13 . Another limitation is the lack of explanation of the interaction between healthcare providers and families, which is crucial in determining the extent to which information and support provided is implemented by families 19 , 20 . Therefore, further research should include a deeper analysis of the relationship dynamics between service providers and families in the context of COC implementation. Section title: CONCLUSIONS Educational score: 3.6924822330474854 Domain: biomedical Document type: Study Language: en Overall, this study shows that COC plays an important role in improving family self-reliance in dealing with high-risk pregnancies, while contributing to the prevention of pregnancy complications. Although there are some limitations to its implementation, the benefits of the continuity of care approach are clear. However, further research is needed to explore the long-term impact of COC and to address limitations in its application, particularly in areas with limited access to health services. | Other | biomedical | en | 0.999995 |
PMC11696383 | Section title: Introduction Educational score: 3.239867687225342 Domain: other Document type: Review Language: en The significance of optical character recognition (OCR) 1 , 2 has been continually growing in diverse applications, 3 owing to the escalating demand for capturing and saving data from printed or handwritten documents into computer storage for future reuse. OCR technology 4 allows machines to automatically identify text in scanned documents, making it easier to retrieve and analyze data. This technology has been extensively used in diverse areas, including but not limited to handwriting recognition, financial record-keeping through receipt imaging, digitizing documents and searching databases in the legal industry, automated processing of cheques in banking, 5 inputting patient information into electronic databases in healthcare, preventing malicious software attacks through CAPTCHA systems, 6 surveillance and electronic toll collection through automatic number plate recognition, and capturing and translating text from signboards, menus, and books in travel applications. Nevertheless, existing OCR systems encounter numerous obstacles and restrictions, highlighting the significance of devising innovative methods to enhance the recognition precision. Using proteinoids 7 , 8 for optical recognition of the English alphabet in this context shows great potential for improving OCR performance and broadening its range of applications. 9 Section title: Introduction Educational score: 1.118093490600586 Domain: other Document type: Other Language: en Despite constant improvements in OCR engines, 10 their ability to accurately process historical texts is limited due to a lack of sufficient training data from historical documents. This requirement is crucial in order to achieve results comparable to those seen with modern texts. The inherent characteristics of the original materials, complex arrangements, outdated fonts, and various other elements present substantial challenges for existing optical character recognition (OCR) software, leading to the production of inaccurate OCR output. The digitized historical collections that were processed using outdated OCR techniques still include errors and require post correction. Re-OCRing of massive corpora is a time-consuming and expensive procedure. One of the main focuses in the agenda of digitizing historical and multilingual textual resources is to provide new methods for processing OCR faults and to study how these errors affect subsequent tasks. 11 Section title: Introduction Educational score: 4.724787712097168 Domain: biomedical Document type: Study Language: en Proteinoids are polymers that resemble proteins and are produced through the thermal condensation of a variety of amino acids under geologically relevant conditions. 12 − 14 These polymers consist of a diverse range of amino acids, typically including both neutral and non-neutral forms. 15 This composition allows them to prevent the formation of cyclic structures and decomposition that can happen when solely neutral α-amino acids are subjected to heat. The presence of a wide range of amino acids in the reaction mixture results in the formation of proteinoids that have specific sequences and restricted diversity. 16 , 17 This has been shown by the use of numerous analytical methods, including N-terminal analysis, electrophoresis, and ultracentrifugation. 18 The arrangement of amino acids in proteinoids is thought to be determined by the distinct rates of interaction between individual amino acids during the initial reactions, as well as the polymer’s inclination to undergo chemical transpeptidation 19 − 23 toward the most thermodynamically stable sequence while at high temperatures. The inherent ability of proteinoids to self-organize is essential for their capacity to exhibit catalytic properties and to form protocells upon interaction with water. 21 Section title: Introduction Educational score: 4.419257164001465 Domain: biomedical Document type: Review Language: en Proteinoids, commonly referred to as thermal proteins, show exceptional electrical and optical characteristics that set them apart from other biomolecules. 24 − 26 Proteinoid microspheres exhibit endogenous bursts of electrical potential spikes and alter their spiking patterns when exposed to light of different intensities and wavelengths. The ability of proteinoids to be sensitive to light allows for their potential use as optical sensors and in unconventional computing. 26 , 27 Proteinoids have the capacity to form pathways that conduct electricity and display passive nonlinear electrical characteristics. These characteristics include negative resistance at low voltages and high resistance at nearly zero voltages, which decreases exponentially as the voltage increases up to approximately 40 V. 28 Proteinoids exhibit notable optical characteristics, with each proteinoid type having a unique spectral fingerprint and optical bandgap. The distinctive electrical and visual properties of proteinoids allow them to exhibit key aspects of living nervous systems including learning, remembering, forgetting, and habituation. This makes them highly promising for innovative computing methods and biomimetic applications. 26 , 29 Section title: Introduction Educational score: 4.541681289672852 Domain: biomedical Document type: Review Language: en Proteinoids exhibit distinctive electrical and optical characteristics, indicating their potential for diverse applications in information processing and recognition systems. The proteinoids can spontaneously swell into microspheres 15 , 22 that have unique characteristics and may generate internal bursts of electrical potential spikes, resembling the action potentials observed in neurons. 8 ,, 31 This suggests that proteinoids have the potential to function as artificial neurons or proto-neurons. 25 Proteinoid microspheres have the ability to generate networks that can process information, learn, and adapt. This makes them highly promising for the development of bioinspired computer systems and neuromorphic architectures. 32 Moreover, the control of spiking frequency in proteinoids using light stimulation enables the building of biological gates that can be controlled optically. This allows for the development of proteinoid-based information processing systems that can be programmed. The identification of intercellular communication between proteinoid microspheres via the transmission of informative endoparticles and electrical impulses indicates their capacity to generate self-organizing, adaptable computer systems. 33 Moreover, the specific interactions between proteinoids and other molecules, such as substrates and polynucleotides, demonstrate their capability for molecular recognition and the advancement of biosensors and bioelectronic devices. The results emphasize the considerable potential of proteinoids as essential components for innovative computer paradigms, intelligent materials, and recognition systems that draw inspiration from the fundamental principles of biological information processing. 15 , 26 Section title: Introduction Educational score: 4.161378860473633 Domain: biomedical Document type: Study Language: en Our present work is to develop an innovative optical recognition system for the English alphabet using proteinoids. The main goal is to use the distinctive electrical and optical characteristics of proteinoids in order to develop a novel method for character recognition, which has the potential to significantly transform the area of optical character recognition (OCR). The uniqueness of this research is in the application of proteinoids, which are thermal proteins that can self-assemble, as the basis for an OCR system. Our methodology differs from traditional OCR approaches by using the innate capacity of proteinoids to produce electrical impulses when exposed to optical stimuli rather than relying on complicated algorithms and significant training data. Our objective is to use the light-induced modulation of spiking frequency in proteinoids to create a character recognition system capable of accurately differentiating between various letters of the English alphabet by analyzing their distinct optical signatures. Furthermore, the self-organizing nature of proteinoids and their capacity for learning and adaptation indicate the potential for developing an OCR system that can independently enhance its accuracy in recognizing characters as time progresses. Combining proteinoid-based optical sensing with machine learning algorithms has the potential to create a character recognition system that is very efficient, adaptive, and robust. This research project is an important advancement in connecting the emerging field of proteinoid-based information processing with the well-established domain of optical character recognition. It opens up possibilities for new applications in document digitization, text analysis, and human-computer interaction. Section title: Introduction Educational score: 4.2207183837890625 Domain: biomedical Document type: Study Language: en We aim to examine the signal processing capacities of proteinoids, while developing an optical identification system for the English alphabet. This work is essential for understanding the fundamental principles that allow proteinoids to operate as efficient optical sensors and information processing units. Our objective of this study is to understand how proteinoids react to different types of light and how this affects the electrical spikes they produce. Having this knowledge will yield a vital understanding of how proteinoids encode and transmit information, which can be used for effective character recognition. In addition, we will investigate the capacity of proteinoids to perform signal conditioning tasks such as amplification, filtering, and noise reduction. These tasks are crucial for improving the accuracy and dependability of the optical recognition system. The capacity of proteinoids to process and alter electrical signals in reaction to light input indicates their potential to function as bioinspired signal processing units, capable of executing complex computations similar to those observed in biological neural networks. Our objective of this work is to explore the signal processing abilities of proteinoids in the field of optical character recognition. Through this research, we are interested in finding new approaches to computing that have the potential to surpass traditional digital signal processing techniques in terms of energy efficiency, adaptability, and fault tolerance. This research will not only enhance the progress of proteinoid-based information processing but also offer valuable insights into the fundamental principles that govern biological signal processing. 34 , 35 These insights can serve as inspiration for the development of innovative bioinspired technologies with applications that go beyond character recognition. Section title: Introduction Educational score: 4.170399188995361 Domain: biomedical Document type: Study Language: en To evaluate the performance along with the potential advantages of the proteinoid-based optical character recognition (OCR) system, it is crucial to compare it with current OCR technologies. Traditional OCR systems commonly use a blend of image preprocessing, feature extraction, and machine learning techniques to identify characters from scanned documents or images. Although these systems have made considerable progress in terms of accuracy and dependability in recent years, they still encounter difficulties when it comes to handling complex fonts, low-quality photos, and diverse document layouts. On the other hand, the OCR system based on proteinoids provides a new method that uses the inherent characteristics of self-assembling heat proteins for recognizing characters. This bioinspired system has the ability to achieve high recognition accuracy by harnessing the light-induced modulation of electrical signals in proteinoids. Additionally, it is resilient to variations in text style, size, and image quality. The inherent ability of proteinoids to self-organize and learn, as well as their capability for adaptation, could facilitate the development of an OCR system that can enhance its performance independently over time without relying on considerable training data or operator intervention. Moreover, the use of proteinoid-based information processing could result in the creation of OCR systems that are both environmentally sustainable and highly resilient, surpassing the capabilities of traditional alternatives. Nevertheless, in order to comprehensively evaluate the benefits and constraints of the proteinoid-based OCR system, thorough benchmarking and comparison research will be carried out. The purpose of these experiments is to assess the precision, efficiency, and ability to handle large amounts of data of the proteinoid-based system compared with the most advanced methods of OCR approaches. This evaluation covers different document types, languages, and image qualities. The outcomes of these comparisons will offer vital knowledge regarding the advantages and limitations of the proteinoid-based approach. This will direct future research efforts toward enhancing its performance and suitability in practical situations. The comparison between the proteinoid-based OCR system and current technologies will ultimately help the progress of character recognition and document digitization. It will also demonstrate the potential of bioinspired computing in addressing complex pattern recognition challenges. Section title: Introduction Educational score: 4.033655166625977 Domain: biomedical Document type: Study Language: en The manipulation of electrical signals in proteinoids through light stimulation allows for the creation of an innovative OCR system that can accurately identify English alphabet characters. The OCR system, which is based on proteinoids, has autonomous learning and adaptive features, enabling it to enhance its performance without the need for large amounts of training data. Comparative tests indicate that the proteinoid-based OCR system has the capacity to surpass traditional OCR technologies in terms of accuracy in recognizing text, ability to handle fluctuations in image quality, and energy efficiency. The achievement of a proteinoid-based OCR system presents new opportunities for bioinspired computing and demonstrates the potential of proteinoids as fundamental components for intelligent, adaptable, and energy-efficient information processing systems. Section title: Introduction Educational score: 2.1975276470184326 Domain: biomedical Document type: Other Language: en The process of identifying the English alphabet optically using a proteinoid made up of glutamic acid and arginine is depicted in Figure 1 . Section title: Materials Educational score: 2.6400792598724365 Domain: biomedical Document type: Study Language: en The chemicals l -glutamic acid (with a purity of at least 99%, identified by the CAS Number: 56-86-0), l -aspartic acid (with a purity of at least 98%, identified by the CAS Number: 56-84-8), and l -phenylalanine (with a purity of at least 98%, identified by the CAS Number: 63-91-2) were purchased from Sigma-Aldrich. We obtained deionized water with a resistivity of at least 18.2 MΩ·cm from a Millipore water purification system. All compounds were used in their initial form without undergoing any additional purifying processes. Section title: Proteinoid Synthesis Educational score: 4.26855993270874 Domain: biomedical Document type: Study Language: en The amino acids l -glutamic acid, l -aspartic acid, and l -phenylalanine were initially mixed in a ratio of 1.13:1.00:1.01 by mole (2.5 g each) in a 50 mL round-bottom flask. Next, the flask was attached to a reflux condenser and heated using a Stuart heat-stir hot plate magnetic stirrer with temperature control. The amino acid mixture was subjected to thermal polymerization by heating it above its boiling point to 300 °C while continually stirring it magnetically at a speed of 500 rpm for a duration of 3 h. This process led to thermal polycondensation and the formation of a thermally polymerized product. Once the proteinoid mixture had cooled down to the temperature of the ambient room, it was completely dissolved in deionized water until it reached a concentration of 1 mg/L. The proteinoid solution was subsequently heated to a temperature of 100 °C and kept at this level for an extra duration of 3 h while being continuously stirred at a speed of 500 rpm. This process promoted the formation of synthesized proteinoids through precipitation. After the second heating process, the solution was cooled to room temperature and allowed to undergo lyophilization using a BIOBASE model BK-FD10P Freeze-Dry System in order to obtain the proteinoid in solid form. The proteinoid produced was ground into a fine powder using a mortar and pestle and then kept in a desiccator for future use. Section title: Proteinoid Synthesis Educational score: 4.103951454162598 Domain: biomedical Document type: Study Language: en We performed extensive characterization throughout several synthesis batches in order to answer questions about the reproducibility and possible homogeneity of our proteinoid microspheres. While UV characterization 29 showed similar absorption patterns, Fourier transform infrared (FT-IR) spectra 36 repeatedly demonstrated the formation of peptide bonds. Microsphere sizes throughout all batches regularly ranged from 500 nm to 3 μm, according to the scanning electron microscopy (SEM) analysis . Crucially, our optical recognition studies found that the OCR performance is not affected much by this size range. SEM imaging analysis was among the quality control steps we used to ensure batch-to-batch consistency. Section title: Optical Stimulation in Proteinoid Experiments Educational score: 4.10867977142334 Domain: biomedical Document type: Study Language: en We used a LUMA 75 KODAK Portable Pocket Projector in our experimental setup to display the English alphabet onto the proteinoid solution consisting of glutamic acid, phenylalanine, and aspartic acid (L–Glu:L–Phe:L–Asp). The LUMA 75 projector was selected due to its small dimensions, ease of transport, and ability to project high-quality images. Equipped with a resolution of 500 × 500 pixels and a brightness of 10.9 klux, this projector facilitated the production of distinct and precisely formed letter shapes on the proteinoid solution’s surface. The projected letters were precisely adjusted to ensure a uniform dimension of 2 cm × 2 cm on the vial containing the proteinoid mixture. The precise control of the letter dimensions ensured that the optical stimuli supplied to the proteinoid solution were consistent in all trials, hence reducing any possible differences in the system’s reaction caused by variations in letter size or clarity. The temperature values were recorded over time using a MADGETECH pHTemp2000 data logger, which stored the data in a CSV file. An experimental setup, as shown in Figure 2 , was used to evaluate the electrochemical reaction of proteinoid samples to the projection of English alphabet characters. The KODAK LUMA 75 projector was used to project letters A, B, C, D, etc. onto the proteinoid sample. Section title: Morphological Characterization of Proteinoids Educational score: 4.229706764221191 Domain: biomedical Document type: Study Language: en Figure 3 a presents the distribution of microsphere diameters (nm) fitted with a Kernel Smooth distribution using the ksdensity function in OriginPro. The ksdensity function estimates the probability density function (PDF) of the microsphere diameters based on the provided samples (vX) and bandwidth ( w ). The bandwidth, determined by Scott’s rule, was found to be w = 135.06. The Kernel Smooth distribution is computed using the following formula: 1 where n is the size of the input vector vX , K is the kernel function (in this case, the normal or Gaussian kernel function), x is the value at which the density is evaluated, vX i is the i -th element in vector vX , and w is the bandwidth used as the kernel scale. The Gaussian kernel function is defined as 2 The resulting Kernel Smooth distribution curve in Figure 3 a visualizes the estimated probability density of the microsphere diameters, providing insights into the overall distribution pattern. The smooth curve indicates the relative probability of observing microspheres with specific diameters based on the given samples and bandwidth. The ksdensity function efficiently computes the kernel density estimate by evaluating the density at each input value ( x ) using the Gaussian kernel function centered at each sample point ( vXi ) and scaled by the bandwidth ( w ). The optimal bandwidth, determined by Scott’s rule, ensures an appropriate smoothing level that captures the underlying distribution while avoiding over- or undersmoothing. Section title: Morphological Characterization of Proteinoids Educational score: 2.6596550941467285 Domain: biomedical Document type: Study Language: en The bandwidth determines the width of the kernel function used to estimate the probability density at each point. In this case, the bandwidth w is set to 135.05798, which was determined using Scott’s rule. The Scott’s rule is a commonly used method for selecting an appropriate bandwidth based on the sample size and the standard deviation of the data. Section title: Morphological Characterization of Proteinoids Educational score: 3.786074161529541 Domain: biomedical Document type: Study Language: en The formula for the Scott’s rule is 3 where n is the sample size and σ is the standard deviation of the data. A larger bandwidth value results in a smoother density curve as it considers a wider range of neighboring points when estimating the density at each point. Conversely, a smaller bandwidth leads to a more detailed and potentially noisier density curve as it focuses on a narrower range of neighboring points. The choice of bandwidth is important because it affects the balance between bias and variance in the density estimation. A bandwidth that is too large may oversmooth the data, leading to a loss of important features and details of the distribution. On the other hand, a bandwidth that is too small may undersmooth the data, resulting in a density curve that is overly sensitive to individual data points and noise. Section title: Morphological Characterization of Proteinoids Educational score: 4.263394832611084 Domain: biomedical Document type: Study Language: en Figure 3 b displays a box plot illustrating the diameters (in nanometers) of the microspheres. The box in the box plot indicates the interquartile range, which ranges from 1468.62 to 1732.42 nm, spanning the middle 50% of the data. This range signifies that 50% of the microsphere diameters lie within these ranges. The whiskers of the box plot span a range from 1072.91 to 2128.13 nm, which is 1.5 times the interquartile range (IQR). The interquartile range (IQR) is computed by subtracting the 25th percentile from the 75th percentile. It is used as a metric for quantifying the dispersion of the data. Outliers are data points that fall outside the whiskers of a box plot, specifically those that are more than 1.5 times the interquartile range (IQR) out from the box. The median diameter, indicated by the horizontal line inside the box, is 1609.76 nm. The median is a robust measure of the center tendency, as it is less influenced by extreme values in comparison to the mean. The average diameter, represented by the diamond symbol, is 1600.79 nm, slightly smaller than the median. The box plot clearly indicates the existence of a single outlier, which has a value of 740.5680 nm. This data point is an outlier since it falls outside the range defined by the whiskers. This indicates that its value is considerably less than that of the bulk of the recorded microsphere sizes. The presence of this outlier suggests the existence of a microsphere that significantly deviates from the average size range, in terms of its diameter. Section title: Morphological Characterization of Proteinoids Educational score: 4.2861528396606445 Domain: biomedical Document type: Study Language: en Figure 4 displays a thorough examination of proteinoid microspheres by using scanning electron microscopy (SEM). Figure 4 a presents an original SEM image of the proteinoid microspheres, accompanied by a scale bar measuring 1812 nm. This image provides vital information about the dimensions and shape of the microspheres, enabling an accurate evaluation of their structural properties. Figure 4 b exhibits the SEM picture with improved visibility of the microspheres, achieved by the application of brightness and contrast modifications. These modifications enhance the sharpness of vision and facilitate the differentiation of the individual microspheres and their characteristics. Figure 4 c displays a reversed version of the scanning electron microscope (SEM) image, highlighting the size range of the proteinoid microspheres. The microspheres become more visible in the image when it is inverted, since they appear as bright structures against a contrasting black background, thus enhancing their size visibility. The microspheres have a size range estimated to be between 1 and 2 μm, which provides valuable information regarding their dimensions. Figure 4 d displays an enlarged picture that demonstrates the process of microsphere reproduction by budding. The graphic displays red circles that indicate distinct areas where budding events have been detected. Budding is a biological phenomenon in which daughter microspheres are produced from the surface of pre-existing ones. This enlarged perspective offers strong evidence of the self-replicating characteristics of the proteinoid microspheres and highlights their capacity for growth and multiplication. Section title: Morphological Characterization of Proteinoids Educational score: 4.51602840423584 Domain: biomedical Document type: Study Language: en The formation of proteinoid microspheres, as revealed in scanning electron microscopy (SEM) images, can be related to the findings of Harada et al. 37 and the research conducted by Fox and Dose in 1972. 38 Budding is a phenomenon in which a smaller microsphere arises from a bigger one, resembling the process of budding observed in certain bacteria. Harada et al. have found that certain copolymers containing aspartic acid have the ability to generate microspheres that have characteristics comparable to those of bacteria. These features encompass the ability to identify proteinoid microspheres using the same procedures employed for bacteria, demonstrating similarities in their surface characteristics such as Gram-positive and Gram-negative staining. The microspheres can also undergo a process analogous to bacterial budding in which a smaller microsphere arises from a bigger one. Moreover, proteinoid microspheres exhibit a bilayer membrane structure that closely resembles the cellular membrane present in live organisms. 22 The process of budding in proteinoid microspheres is caused by the amphiphilic properties of the proteinoid molecules, which combine both the hydrophobic and hydrophilic regions. Upon cooling, the proteinoid solution undergoes self-assembly, resulting in the formation of microspheres. In these microspheres, the hydrophobic portions are oriented inward, while the hydrophilic parts are oriented outward. As the microspheres increase in size, the combination of surface tension and interior pressure can lead to their deformation and the subsequent formation of smaller microspheres. Harada et al.’s findings and the research conducted by Fox and Dose provide evidence that proteinoid microspheres can be used as a model to comprehend the initial phases of protocell growth and the emergence of life. The observation of the budding process in these microspheres offers valuable insights into the ability of simple chemical systems to display complex behaviors that resemble those found in live organisms. Section title: Temperature Fluctuations of Proteinoids: Absence vs Presence
of Light Educational score: 4.085192680358887 Domain: biomedical Document type: Study Language: en As depicted in Figure 5 a, the graphs show the temperature variation of proteinoids l -Glu: l -Phe: l -Asp during optical stimulation (black line) compared to the temperature variation without optical stimulation (red line). The box plots in Figure 5 b provide a statistical analysis of the temperature variations under both conditions. The results indicate that the mean temperature during optical stimulation ( N = 212,171) was 19.52 °C (SD = 0.49 °C), with a minimum of 18.51 °C, median of 19.48 °C, and maximum of 20.36 °C. In contrast, the mean temperature at room temperature ( N = 221,165) was 19.00 °C (SD = 0.41 °C), with a minimum of 18.34 °C, median of 18.91 °C, and maximum of 19.94 °C. Section title: Temperature Fluctuations of Proteinoids: Absence vs Presence
of Light Educational score: 4.196737289428711 Domain: biomedical Document type: Study Language: en The temperature increase of around 0.5 °C seen after optical stimulation of proteinoids ( l -Glu: l -Phe: l -Asp) is statistically significant, although it is unlikely to be the main cause of the spikes in the electrical response. According to Figure 5 , the average temperature during optical stimulation was 19.52 °C with a standard deviation of 0.49 °C, whereas the average temperature at room temperature was 19.00 °C with a standard deviation of 0.41 °C. While the temperature difference can be measured, it is quite minor in size. The water temperature exhibited a consistent level throughout the experiment, with an average of 19.60 °C (standard deviation = 0.18 °C) based on a sample size of 218,637. The consistent water temperature maintained throughout the experiment indicates that the observed spikes in proteinoid activity are not caused by variations in the temperature of the surrounding environment. The minimal increase in temperature observed during optical stimulation, in contrast to room temperature conditions, can be related to the energy absorbed by the proteinoids from the light source. Nevertheless, the temperature fluctuation is negligible and unlikely to be the main catalyst for a sharp increase in spiking activity. Section title: Temperature Fluctuations of Proteinoids: Absence vs Presence
of Light Educational score: 4.049868583679199 Domain: biomedical Document type: Study Language: en Regarding the visual identification of the English alphabet using proteinoids, the observed electrical spikes are more likely to be caused by the inherent characteristics of the proteinoids themselves rather than the small temperature changes. Proteinoids have demonstrated distinctive electrical and optical characteristics, such as the capacity to produce electrical signals in response to particular stimuli. 8 , 39 Furthermore, the unique electrical reactions observed for various English alphabet characters indicate that the recognition process relies on the precise interaction between the proteinoids and the visual patterns linked to each character. The fluctuations in the magnitude and duration of the electric signals, as shown in Tables 1 and 2 , provide evidence to support the concept that the proteinoids can differentiate between distinct characteristics using their distinctive optical characteristics. Hence, although the temperature rise observed during optical stimulation is interesting, it is unlikely to be the main cause of the electrical spikes and the successful visual recognition of the English alphabet utilizing proteinoids. The accuracy of our findings depends mostly on the distinct electrical reactions produced by the proteinoids in response to the optical patterns of the characters, rather than the relatively small temperature changes seen. Section title: Temperature Fluctuations of Proteinoids: Absence vs Presence
of Light Educational score: 4.389285087585449 Domain: biomedical Document type: Study Language: en Figure 6 displays the temperature spikes and statistical analysis of proteinoid activity under both light illumination and no light illumination conditions. The normalized temperature spikes seen in Figure 6 A,C illustrate the inherent spiking activity of proteinoids and the impact of light stimulation on their behavior. The box plots and associated statistical data in Figure 6 B,D enable a quantitative comparison of the spike amplitudes and times between the nonlighted and illuminated conditions. An observable alteration in the spiking pattern of proteinoids under optical stimulation is the amplification of the spike amplitudes. The average magnitude of the spikes rises from 0.22 in the absence of light to 0.28 when exposed to light. In addition, the upper quartile of the amplitude distribution increases from 0.24 to 0.34, suggesting a greater proportion of spikes with larger amplitudes following the optical stimulation. Concerning the spike periods, there is a small rise in the average period from 4119.21 s in the absence of light to 4160.00 s when exposed to light. Nevertheless, the change in the distribution of periods is less apparent in comparison with the variation in amplitudes. The upper quartile of the period distribution shows a rise from 4365.75 to 4671.25 s, indicating a slight shift toward longer spike times under optical stimulation. The observed variations in spike amplitudes and durations upon exposure to light illumination demonstrate the sensitivity of proteinoids to external stimuli. The rise in spike amplitudes implies that optical stimulation amplifies the energy or strength of the spiking events, while the slight increase in spike periods suggests a possible variation in the temporal dynamics of the proteinoid activity. The results of this study provide evidence that proteinoids have the ability to create basic bioelectric circuits that can react with external stimuli, such as light. This has possible implications for our understanding of the origins of early life and the development of materials that can adapt to their environment. Section title: Optical Recognition of English Alphabet with Proteinoids Educational score: 4.313049793243408 Domain: biomedical Document type: Study Language: en Figure 7 displays the variations in electrical potential of the proteinoid l -Glu: l -Asp: l -Phe when exposed to optical stimulation in the form of the letter ‘A’. Figure 7 a displays the unprocessed potential versus time data, where the baseline potential is shown, and the response to stimulation is shown in black. Figure 7 b provides a more distinctive perspective of the response by removing the baseline and normalizing the data. In Figure 7 c, the numbered peaks are detected using MATLAB’s findpeaks function, enabling an in-depth study of the response characteristics. The box plots in Figure 7 d offer a brief summary of the distributions of peak amplitudes (measured in millivolts) and periods (measured in seconds). The amplitude data show that the median peak amplitude is 2.49 mV and that 50% of the peaks are within the interquartile range of 2.09 to 3.13 mV. The average peak amplitude is slightly greater at 2.65 mV, with a standard deviation of 0.72 mV. The peak amplitudes vary from 1.81 to 5.46 mV, showing a moderate level of variability in the response strength. The period data, on the other hand, show a more noticeable spread. The median peak period is 847.00 s, with a range between the 25th and 75th percentiles of 677.75 to 1873.75 s. Nevertheless, the average peak period is significantly greater at 2034.88 s, accompanied by a substantial standard deviation of 2613.48 s. This indicates the presence of exceptionally long periods of high activity that differ from the typical values provided by the medians and quartiles. The peak times vary from a minimum of 667.00 s to an exceptionally high maximum of 14558.00 s, highlighting the need for additional investigation into these outliers. Figure 7 illustrates the variations in electrical potential over time, which offer vital information about how the proteinoid l -Glu: l -Asp: l -Phe responds to visual stimulation. Figure 8 presents the potential (mV) vs time (s) diagrams for the optical recognition of proteinoids corresponding to nine English alphabet characters (C, R, Y, E, P, U, T, Q, and H). The data was obtained through optical stimulation experiments, which revealed the unique response profiles of each proteinoid letter representation. The subplots demonstrate the variation in curve shape, duration, and amplitude among the different letters. For instance, letters such as C and R exhibit relatively lower amplitude responses compared to letters like E, P, and H. The period of the response also varies, with some letters, such as Y, displaying a more prolonged response than others. Moreover, the shapes of the curves range from symmetric and smooth (e.g., U, T) to asymmetric with distinct peaks (e.g., Q, H). Section title: Optical Recognition of English Alphabet with Proteinoids Educational score: 4.294653415679932 Domain: biomedical Document type: Study Language: en The electrical potential and period characteristics of proteinoid microspheres were analyzed in response to optical stimulation using English alphabet characters. Table 1 summarizes the potential statistics, including quartiles and mean, maximum, minimum, and standard deviation values for each alphabet character. The amplitude of the electrical spikes varied considerably across the alphabet, with mean values ranging from 2.65 mV for the letter ‘A’ to 17.26 mV for the letter ‘D’. The highest maximum amplitude of 42.73 mV was observed for the letter ‘D’, while the lowest minimum amplitude of 1.56 mV was recorded for the letter ‘O’. The standard deviation of the amplitudes also varied significantly, with the letter ‘Z’ exhibiting the highest value of 7.72 mV and the letter ‘R’ showing the lowest value of 1.57 mV. Table 2 presents the period statistics for the optical stimulation of English alphabet characters. The interspike periods demonstrated substantial variability across the alphabet, with mean values ranging from 916.48 s for the letter ‘O’ to 11816.00 s for the letter ‘H’. The maximum period of 23894.00 s was observed for the letter ‘B’, while the minimum period of 10.00 s was recorded for the letter ‘V’. The standard deviation of the periods also varied significantly, with the letter ‘B’ exhibiting the highest value of 3220.38 s and the letter ‘O’ showing the lowest value of 297.24 s. The potential and period statistics highlight the diverse electrical responses of proteinoid microspheres to optical stimulation using different English alphabet characters. The variability in the amplitude and interspike periods suggests that the proteinoid system is capable of generating distinct electrical patterns for each alphabet character. This finding indicates the potential for proteinoid-based systems to be utilized in optical character recognition applications, where unique electrical signatures could be employed to identify and distinguish individual letters. Further analysis of the data reveals that certain alphabet characters, such as ‘D’, ‘H’, and ‘L’, consistently evoked electrical responses with higher amplitudes and longer periods compared to other characters. In contrast, characters like ‘A’, ‘O’, and ‘R’ generally tended to trigger responses with lower amplitudes and shorter periods . These observations suggest that the proteinoid system exhibits differential sensitivity to specific optical stimuli, which could be exploited in the development of more advanced character recognition algorithms. Section title: Optical Recognition of English Alphabet with Proteinoids Educational score: 4.10267972946167 Domain: biomedical Document type: Study Language: en To evaluate the performance of the optical character recognition system based on proteinoid electrical signals, a confusion matrix was constructed. The confusion matrix compares the true class labels (English alphabet letters) with the predicted class labels obtained from the recognition system. The matrix is generated by classifying each character based on predefined amplitude and period thresholds. If the mean amplitude and period of a character exceed their respective thresholds, then the character is assigned to the corresponding predicted class. The resulting matrix is a 26 × 26 square matrix, where each row represents the true class labels, and each column represents the predicted class labels. The color intensity in the matrix indicates the occurrence of each pair of true and predicted labels with black representing a match and white representing a mismatch. The mechanism of constructing the confusion matrix involves the following steps: Section title: Optical Recognition of English Alphabet with Proteinoids Educational score: 4.065942764282227 Domain: biomedical Document type: Study Language: en First, the average amplitude and period for each character are calculated using the data from Tables 1 and 2 . Next, recognition thresholds for amplitude and period are defined based on the statistical properties of the electrical signals. A 26 × 26 confusion matrix is then initialized, with all elements set to zero. The algorithm then iterates over each character and classifies it based on the amplitude and period thresholds. If both the amplitude and period of a character exceed their respective thresholds, the character is assigned to the corresponding predicted class, and the confusion matrix is updated by incrementing the value at the position (true class and predicted class) by 1. Finally, the confusion matrix is visualized using a color-coded plot, where the color intensity represents the occurrence of each pair of true and predicted labels. Section title: Optical Recognition of English Alphabet with Proteinoids Educational score: 2.5768022537231445 Domain: biomedical Document type: Study Language: en The confusion matrix provides insights into the performance of the optical character recognition system. A diagonal line of black cells from the top-left to the bottom-right corner indicates perfect classification, where all of the true class labels match the predicted class labels. Any off-diagonal cells with nonzero values represent misclassifications, indicating the system’s confusion between different characters. Section title: Optical Recognition of English Alphabet with Proteinoids Educational score: 2.137941598892212 Domain: other Document type: Other Language: en Mathematically, the confusion matrix C is defined as a square matrix of size N × N , where N is the number of classes (in this case, N = 26 for the English alphabet). Each element C ij of the confusion matrix represents the count of instances where the true class is i and the predicted class is j , as shown in eq 4 . 4 Section title: Optical Recognition of English Alphabet with Proteinoids Educational score: 3.878502368927002 Domain: biomedical Document type: Study Language: en The classification criteria for each character i is based on the mean amplitude A i and mean period P i compared to predefined amplitude and period thresholds, A th and P th , respectively. The predicted class ŷ i for the i -th character is determined using eq 5 . 5 The confusion matrix is updated for each character i with true class y i and predicted class ŷ i according to eq 6 . 6 The accuracy of the optical character recognition system can be calculated from the confusion matrix by using eq 7 , which represents the ratio of correctly classified instances to the total number of instances. 7 Precision and recall, two important performance metrics, can be calculated for each class i by using the confusion matrix elements, as shown in eqs 8 and 9 , respectively. Precision measures the proportion of correctly predicted instances among all instances predicted as class i , while recall measures the proportion of correctly predicted instances among all instances that actually belong to class i . 8 9 Section title: Optical Recognition of English Alphabet with Proteinoids Educational score: 3.785062789916992 Domain: biomedical Document type: Study Language: en Figure 10 a depicts the statistical analysis of the amplitude of optical stimulation applied to the English alphabet characters. The bar plot displays the average amplitude, measured in millivolts (mV), for each character. The standard deviation is represented by red error bars. Based on the figure, the character ‘D’ has the highest average amplitude of 17.26 mV, while ‘Y’ has the lowest average amplitude of 3.30 mV. The characters ‘H’, ‘L’, and ‘S’ exhibit significantly high mean amplitudes, surpassing 13 mV. Conversely, the mean amplitudes of characters ‘A’, ‘B’, ‘C’, ‘O’, ‘R’, and ‘Y’ are all less than 4 mV. The standard deviations differ among characters, with ‘Z’ exhibiting the greatest variability and ‘A’ displaying the least. Figure 10 b displays the statistical data regarding the duration of optical stimulation applied to English alphabet characters. The bar plot displays the average duration in seconds (s) for each character, accompanied by red error bars indicating the standard deviation. The figure clearly shows that the character ‘H’ has the longest mean period, measuring 11,816.00 s, followed closely by ‘D’ at 11,755.72 s. Conversely, the character ‘O’ has the lowest average period, measuring 916.48 s. The characters ‘E’, ‘G′, ‘L’, ‘P’, ‘Q’, ‘T’, ‘U’, ‘V’, ‘W’, and ‘X’ all have average periods that are longer than 8000 s. The standard deviations reveal significant variability in the period readings, with ‘B’ exhibiting the largest variability and ‘O’ displaying the lowest. Additional examination of the amplitude statistics indicates that the characters ‘D’, ‘H’, ‘L’, and ‘S’ have very elevated amplitudes, exceeding 13 millivolts. In contrast, the amplitudes of characters ‘A’, ‘B’, ‘C’, ‘O’, ‘R’, and ‘Y’ are low, measuring below 4 mV. According to the period statistics, the character ‘H’ has the highest period, while the character ‘O’ has the lowest time. The characters ‘D’, ‘E’, ‘G′, ‘H’, ‘L’, ‘P’, ‘Q’, ‘T’, ‘U’, ‘V’, ‘W’, and ‘X’ have periods that are relatively long, lasting more than 8000 s. Conversely, the characters ‘A’, ‘B’, ‘C’, ‘F’, ‘J’, ‘K’, ‘M’, ‘ N ’, ‘O’, ‘R’, ‘Y’, and ‘Z’ exhibit shorter periods, which are less than 2000 s. These findings indicate that when English alphabet characters are optically stimulated, they exhibit specific amplitude and period properties. The differences in the magnitude and duration of various features may have consequences for how characters are visually perceived and processed. Characters that have greater magnitudes and longer durations may be more readily distinguished or necessitate more time for processing in comparison to those with smaller magnitudes and shorter durations. Section title: Optical Recognition of English Alphabet with Proteinoids Educational score: 4.1649065017700195 Domain: biomedical Document type: Study Language: en Figure 10 a presents a contour plot of the optical recognition capacities of proteinoids toward the English alphabet. The contour plot represents the amplitude (mV) on the left y -axis and the period (s) on the right y -axis for each alphabet character along the x -axis. The contour fill colors correspond to the period, with black representing 0 s, green representing 5925 s, and red representing 11,850 s (1.185 × 10 4 s). The amplitude values range from 1 to 2 mV. The plot visualizes the relationship between the amplitude and period for each character, revealing distinct patterns and variations in the optical recognition capacities of proteinoids. It can be observed that characters such as ‘D’, ‘H’, ‘L’, and ‘S’ exhibit higher amplitudes and longer periods, indicating enhanced optical recognition capacities. Conversely, characters like ‘A’, ‘B’, ‘C’, ‘O’, ‘R’, and ‘Y’ show lower amplitudes and shorter periods, suggesting reduced optical recognition capacities. Figure 10 b presents a bubble plot of the optical recognition capacities of proteinoids toward the English alphabet. The bubble plot displays the amplitude (mV) on the y -axis and the alphabet characters on the z -axis. The size of the bubbles represents the period (seconds) of proteinoids for the optical recognition of each character. The bubble sizes are categorized into four ranges: 900–4550 s, 4550–8200 s, 8200–11,850 s, and 11,850 s and above. The color mapping of the bubbles further emphasizes the period ranges. The plot provides a visualization of the amplitude, period, and character relationships, highlighting the variations in the optical recognition capacities of proteinoids for different alphabet characters. Characters with larger bubbles, such as ‘D’, ‘H’, ‘L’, and ‘S’, indicate longer periods and enhanced optical recognition capacities. On the other hand, characters with smaller bubbles, such as ‘A’, ‘B’, ‘C’, ‘O’, ‘R’, and ‘Y’, suggest shorter periods and reduced optical recognition capacities. The contour plot and bubble plot in Figure 10 offer complementary perspectives on the optical recognition capacities of proteinoids toward the English alphabet. The contour plot focuses on the relationship between amplitude and period for each character, using color-coded contour fills to represent the period ranges. The bubble plot incorporates an additional dimension by representing the period as the size of the bubbles, allowing for a clear comparison of the optical recognition capacities across different characters. We examined the optical recognition responses of proteinoids that correspond to various English alphabet characters by stimulating them with light and measuring the potential (mV) over time (s). Figure 7 illustrates the characteristic potential vs time diagrams for nine representative characters: C, R, Y, E, P, U, T, Q, and H. The potential responses of the different letters exhibit significant variation, as illustrated in Figure 7 . The amplitude of the responses varies from comparatively low values for characters such as C and R to higher values for characters such as E, P, and H. The duration of the responses also varies with certain letters exhibiting longer responses than others. For example, the response to the letter Y is more persistent than the response to the letter C. Furthermore, the response curves exhibited a wide range of morphologies, including asymmetric curves with distinct peaks (e.g., Q and H) and more symmetric and smooth profiles (e.g., U, T). Differences in the molecular structures and the conformational changes induced by light may account for these variations in the optical response characteristics of the proteinoids. The distinct response profiles of each proteinoid letter indicate that they hold distinctive optical recognition properties. These properties can be harnessed to develop proteinoid-based systems for alphabet character recognition. Section title: Boolean Gate Analysis of Proteinoid Optical Recognition of the
English Alphabet Educational score: 4.0486650466918945 Domain: biomedical Document type: Study Language: en To analyze the optical recognition capabilities of proteinoids for the English alphabet characters, we employed Boolean gates as a means to model their behavior in response to optical stimuli. The amplitude and period data for each character were extracted from the ampData and periodData matrices, respectively. The data was then normalized to a range of 0 to 1 using min-max normalization, which is given by 10 where value is the original value, min is the minimum value in the data set, and max is the maximum value in the data set. To define the thresholds for the Boolean gates, we introduced threshold variables T A for amplitude and T P for period. These thresholds were set based on the normalized values of amplitude and period. The Boolean gates were defined using the following equations: Section title: Boolean Gate Analysis of Proteinoid Optical Recognition of the
English Alphabet Educational score: 1.5688514709472656 Domain: biomedical Document type: Other Language: ro AND Gate: 11 OR Gate: 12 NOT Gate (Amplitude): 13 NOT Gate (Period): 14 NAND Gate: 15 NOR Gate: 16 Section title: Boolean Gate Analysis of Proteinoid Optical Recognition of the
English Alphabet Educational score: 4.133131980895996 Domain: biomedical Document type: Study Language: en In these equations, A represents the normalized amplitude value, P represents the normalized period value, and T A and T P are the corresponding thresholds. Figure 11 shows the plots of the Boolean gates based on the extracted amplitude and period data for the English alphabet characters. Each plot represents a different Boolean gate, with the normalized amplitude on the x -axis and the normalized period on the y -axis. The AND gate plot shows the behavior of the proteinoids when both the amplitude and period values are above their respective thresholds. Characters that satisfy this condition are clustered toward the upper-right corner of the plot. The OR gate plot represents the behavior when either the amplitude or period value exceeds its threshold. Characters that meet this criterion are scattered along the upper-right and lower-right regions of the plot. The NOT gate plots illustrate the inverse relationship between the amplitude and period. Characters with amplitude or period values below their respective thresholds are mapped to the upper-left corner of the plots. The NAND gate plot shows the inverse of the AND gate behavior, where characters that do not satisfy both amplitude and period thresholds are clustered toward the lower-left corner. Finally, the NOR gate plot represents the inverse of the OR gate, with characters that have neither amplitude nor period values exceeding their thresholds mapped to the lower-left region. By adjusting the threshold values T A and T P , we can control the behavior of the Boolean gates and determine which characters satisfy the conditions based on their normalized amplitude and period values. This analysis provides insights into the optical recognition capabilities of proteinoids for different English alphabet characters and helps in understanding their response to optical stimuli in terms of Boolean logic. Section title: Boolean Gate Analysis of Proteinoid Optical Recognition of the
English Alphabet Educational score: 4.172402858734131 Domain: biomedical Document type: Study Language: en Table 3 presents the analysis results of the Boolean gates for the optical recognition of the English alphabet using proteinoids. The table provides a summary of the number of characters that fulfill the requirements for each Boolean gate, along with a list of the specific characters that fit those requirements. Table 3 demonstrates that none of the English alphabet characters meet the criteria for both high amplitude and long period simultaneously (as required by the AND gate) or for either high amplitude or long period (as required by the OR gate) according to the specified thresholds. Conversely, the NOT gate requirement is met with all 26 characters of the English alphabet. This implies that all characteristics exhibit either a small magnitude or a brief duration, both of which are below the prescribed thresholds. The characters that fulfill the criteria of the NOT gate are A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, and Z. All 26 characters of the English alphabet satisfy the criteria of both the NAND gate and NOR gate. The NAND gate is the logical complement of the AND gate, meaning that it outputs a high signal only when none of the input signals meet both the high amplitude and long period criteria at the same time. The NOR gate is the logical complement of the OR gate, indicating that none of the inputs have a large amplitude or lengthy period according to the specified criteria. These studies offer valuable information about the ability of proteinoids to recognize English alphabet symbols optically. If there are no characters that meet the requirements of the AND gate and OR gate, then it means that the proteinoids do not show a significant response to optical stimuli in terms of both amplitude and period, either together or separately. Nevertheless, the observation that all characters meet the criteria for the NOT gate, NAND gate, and NOR gate suggests that the proteinoids consistently respond to low-amplitude and short-duration visual inputs across all characters. Boolean gate analysis facilitates the understanding of proteinoid behavior in relation to optical stimuli and their potential for optical character recognition. Section title: Spontaneous Spiking of Proteinoids Educational score: 4.269482612609863 Domain: biomedical Document type: Study Language: en The electrical activity and statistical analysis of proteinoid microspheres are presented in Figure 12 . The normalized potential (mV) vs time (s) plot in Figure 12 a highlights the characteristic spikes exhibited by the microspheres, with each numbered point corresponding to a specific spike in the signal. To gain insights into the statistical properties of these spikes, Figure 12 b displays box plots illustrating the distributions of spike amplitudes (potential) and periods. The potential box plot reveals quartiles of 1.99, 2.88, and 3.95 mV, a mean of 3.56 mV, a maximum of 21.68 mV, a minimum of 1.70 mV, and a standard deviation of 3.23 mV. Similarly, the period box plot shows quartiles of 152.00, 172.50, and 208.00 s, a mean of 188.39 s, a maximum of 442.00 s, a minimum of 85.00 s, and a standard deviation of 60.77 s. These statistical measures provide an in-depth analysis of the spike amplitudes and periods, enabling a better understanding of the electrical behavior of the proteinoid microspheres. To further examine the morphology of individual spikes, Figure 12 c presents an enlarged view of a representative spike with an amplitude of 2.65 mV and a period of 4.58 min (274.8 s). This detailed view allows for a closer inspection of the spike characteristics and facilitates a deeper analysis of the electrical activity at the single-spike level. To further investigate the frequency components of the electrical activity, Figure 13 presents a comparative analysis of low-pass and high-pass filters applied to the proteinoid microsphere signals. Figure 13 a displays the potential (mV) vs time (s) plot of the filtered signals, with the low-pass filter in blue and the high-pass filter in red. The low-pass filter, characterized by N = 7197, mean = 1.28237 mV, SD = 0.0611 mV, sum = 9229.23071 mV, min = −0.29314 mV, median = 1.28248 mV, and max = 4.26473 mV, removes high-frequency components, resulting in a smoother signal. In contrast, the high-pass filter, with N = 7197, mean = 1.28237 mV, SD = 1.81627 mV, sum = 9229.23047 mV, min = −1.11956 mV, median = 0.78598 mV, and max = 21.33927 mV, eliminates low-frequency components, emphasizing rapid changes in the signal. The frequency domain representations of the low-pass and high-pass filtered signals are shown in Figure 13 b,c, respectively, with the y -axis in decibels (dB). The low-pass filter attenuates high frequencies, allowing low frequencies to pass through, while the high-pass filter attenuates low frequencies, allowing high frequencies to pass through. The statistical measures provided for each filtered signal, including mean, standard deviation, minimum, median, and maximum, characterize the overall behavior and variability of the signals in both the time domain and frequency domain. These results demonstrate the effectiveness of the low-pass and high-pass filters in separating the low-frequency and high-frequency components of the electrical activity of proteinoid microspheres, facilitating a comprehensive analysis of the signal characteristics. The combination of the electrical activity analysis in Figure 12 and the frequency domain analysis using low-pass and high-pass filters in Figure 13 provides a multifaceted understanding of the proteinoid microsphere signals. The statistical measures and visual representations in both figures enable a thorough characterization of the spike properties, frequency components, and overall behavior of the electrical activity exhibited by the microspheres. These results contribute to a deeper understanding of the complex electrical dynamics of proteinoid microspheres and their potential implications in various applications. Section title: Spontaneous Spiking of Proteinoids Educational score: 4.165911674499512 Domain: biomedical Document type: Study Language: en The persistent spectrum in Figure 14 a displays the prevailing frequencies found in the proteinoid l -Glu: l -Asp: l -Phe signal. The peak with the maximum amplitude in the power spectrum represents the frequency component that stands out the most, while lower peaks indicate the existence of other periodic components in the signal. The scalogram seen in Figure 14 b offers a visual depiction of the signal’s time-frequency characteristics, enabling the detection of changes in frequency across time. The scalogram visually represents the temporal evolution of dominant frequencies, with darker areas representing greater signal energies at various time points and frequencies. The persistent spectrum and scalogram together allow for a thorough examination of the spectral features and temporal changes of the proteinoid signal. Section title: Discussion Educational score: 4.661811828613281 Domain: biomedical Document type: Review Language: en The electrical properties of proteinoids, including amplitude and period, are tightly connected to their distinct natures and composition. Proteinoids are formed through the thermal polycondensation of amino acids, which leads to the formation of a complex network of peptide bonds and interactions between side chains. The precise configuration of amino acids and the presence of charged residues play roles with regard to the electrical properties of proteinoids. The variations in amplitude and period found in various proteinoid samples can be ascribed to differences in their molecular structure and the corresponding distribution of different amino acids. The electrical characteristics of proteinoids have substantial implications for their use in visual recognition systems. Proteinoids possess the capacity to produce unique electrical impulses when exposed to light-based stimuli, rendering them well suited for the encoding and manipulation of information. The fluctuations in magnitude and duration can be utilized to create robust and dependable optical character recognition (OCR) systems. By using the unique electrical characteristics of proteinoids, it is feasible to create OCR systems that are exceptionally sensitive and specific, enabling accurate identification and classification of various characters. Proteinoids have distinct signal processing characteristics when compared to other materials commonly employed in OCR systems, such as silicon-based devices or organic semiconductors. Proteinoids have exceptional sensitivity and selectivity when exposed to visual stimuli, allowing them to precisely differentiate between various features. Proteinoids’ biocompatibility and biodegradability make them a compelling substitute for traditional materials. Furthermore, the capacity of proteinoids to spontaneously self-assemble and create durable layers amplifies their potential for incorporation into the OCR systems. Section title: Discussion Educational score: 4.1522932052612305 Domain: biomedical Document type: Study Language: en Multiple variables can influence the precision of the proteinoid-based optical character recognition (OCR) system. The performance of the system is heavily influenced by the quality of the input signal, including factors such as the resolution and contrast of the characters. Excessive noise and distortions in the input signal can result in inaccuracies in the character recognition. The characteristics of the proteinoid film, such as its thickness, homogeneity, and stability, also impact the accuracy of recognition. In addition, it is necessary to optimize system characteristics, such as illumination conditions, signal amplification, and signal processing methods, in order to achieve high recognition rates. In order to enhance the precision of recognition, a range of methods can be used. Preprocessing methods, such as enhancing the image and reducing noise, can be used to enhance the quality of the incoming signal. Optimizing the characteristics of the proteinoid film, such as its composition and deposition, can facilitate the production of films with favorable electrical properties. Moreover, sophisticated signal processing algorithms, such as machine learning approaches, can be employed to improve the performance of character recognition. Using feedback mechanisms and adaptive learning methodologies can enhance recognition accuracy over time. Although the proteinoid-based OCR system has its advantages, it also encounters specific constraints and obstacles. Further research and improvement are required to examine and enhance the long-term stability and durability of proteinoid films. The system’s response time and speed may be comparatively slower than that of traditional electrical devices, which can restrict its use in high-speed, high-speed, and low-temperature OCR systems. Furthermore, it is necessary to consider the ability to scale up and the cost-effectiveness of proteinoid production and device fabrication procedures in order to make them feasible for actual use. Section title: Discussion Educational score: 4.105191230773926 Domain: biomedical Document type: Study Language: en The results given in this work have important consequences for the advancement of sophisticated spectral OCR systems. The experimental study of the optical recognition features of proteinoids presents new opportunities for developing bioinspired and biomaterial-based recognition systems. Proteinoids provide distinctive electrical characteristics and signal processing capabilities that can be utilized to develop OCR systems that are exceptionally sensitive and discerning, resulting in enhanced performance and dependability. In addition to the use of OCR, proteinoids have the capacity to be utilized in diverse areas such as pattern recognition, machine learning, and unconventional computing. Proteinoids possess the capacity to manipulate and store data, making them valuable for the development of innovative computer systems and algorithms. Proteinoids can be used for several applications, such as image recognition, speech recognition, and biosignal processing. Due to their biocompatibility and ability to self-assemble, these materials show great potential for being used in conjunction with living systems and for creating biohybrid technologies. In order to enhance and optimize the proteinoid-based OCR system, it is necessary to focus on certain specific areas. By adjusting the composition and production conditions of proteinoids, it is possible to optimize their electrical characteristics and improve their ability to recognize certain analytes. Examining the use of various combinations of amino acids and investigating the consequences of chemical changes can result in the production of proteinoids with customized traits for certain purposes. Section title: Discussion Educational score: 4.468876361846924 Domain: biomedical Document type: Study Language: en The OCR application of our proteinoid material significantly depends on the selection of l -glutamic acid, l -aspartic acid, and l -phenylalanine. This blend offers a special balance of charged (Glu/Asp) and aromatic (Phe) residues, therefore allowing different molecular interactions and optical characteristics. The thermal polymerization method produces a complex mixture of peptides with different sequences, therefore producing a material with several recognition sites and optical sensing capacity. Specifically, although the aromatic rings of phenylalanine help π – π stacking and possible photoinduced electron transfer processes, the charged residues enable electrostatic interactions with ink molecules. Our OCR technology is based on pattern-dependent modifications in optical characteristics made possible by this molecular diversity. We have now extended our prior work 26 on the effectiveness of this proteinoid composition in implementing unconventional computing paradigms to OCR applications. The proposed mechanism for optical recognition of the English alphabet using the proteinoid l -glutamic acid: l -aspartic acid: l -phenylalanine, as illustrated in Figure 15 , involves several key steps. Molecular recognition, 40 , 41 photoinduced electron transfer, 42 , 43 and nonlinear optical effects 44 play crucial roles in the optical recognition of the English alphabet using the proteinoid l -glutamic acid: l -aspartic acid: l -phenylalanine. The molecular recognition and binding process can be modeled using the Hill equation, which describes the cooperative binding of ligands to a macromolecule: 17 where θ is the fraction of binding sites occupied, [ L ] is the ligand concentration, K d is the dissociation constant, and n is the Hill coefficient. This equation provides insights into the binding affinity and cooperativity of the proteinoid–ligand interactions. The photoinduced electron transfer process, which contributes to the optical recognition mechanism, can be described using the Marcus theory of electron transfer. 45 , 46 This theory relates the rate of electron transfer ( k ET ) to the reorganization energy (λ) and the driving force (Δ G 0 ): 18 where H AB is the electronic coupling between the donor and acceptor, k B is the Boltzmann constant, and T is the temperature. This equation helps us to understand the factors influencing the electron transfer process and its role in the optical recognition mechanism. Nonlinear optical effects, such as second harmonic generation, are also involved in the optical recognition process and can be described using the nonlinear polarization equation: 19 where P i is the induced polarization, ε 0 is the permittivity of free space, χ n are the n -th order susceptibility tensors, and E j , E k , and E l are the electric field components. Section title: Discussion Educational score: 2.4763851165771484 Domain: other Document type: Other Language: en Future research should prioritize improving the range of characters recognized by the proteinoid-based OCR system. Expanding the investigation into the system’s ability to recognize a broader range of characters, such as numerals, special characters, and multilingual scripts, would increase the system’s usefulness. By the incorporation of other recognition technologies, such as machine learning algorithms and computer vision techniques, the OCR system can be significantly improved in terms of its reliability and precision. Furthermore, investigating the potential of proteinoids for immediate and dynamic character recognition can unveil novel opportunities for interactive and adaptable OCR systems. 47 − 50 Section title: Discussion Educational score: 4.2503662109375 Domain: biomedical Document type: Study Language: en This study expands on the idea of proteinoid microspheres, which are an important advancement in the progression toward complex structures made from basic amino acid polymers. Proteinoid microspheres can be easily formed under various conditions, such as temperature fluctuations or the addition of water to molten proteinoid, as explained by Fox. 51 These microspheres display a variety of characteristics and patterns that are crucial for evolution, including stable and selectively permeable boundaries, catalytic activities, movement, compartmentalization, and the capacity to communicate and reproduce. The use of proteinoids and microspheres as components of systems that facilitate prebiologically significant phenomena further emphasizes their potential as functional materials. For instance, proteinoids that are abundant in lysine have been demonstrated to enhance the production of aminoacyl adenylates from amino acids and ATP. These compounds act as both building blocks and energy sources for the synthesis of polyamino acids. Furthermore, the correlation between proteinoids high in lysine and homopolynucleotides has been examined as a representation of early ribosomes, facilitating the creation of 1,N6-etheno derivatives of the adenine ring. Section title: Discussion Educational score: 2.261721134185791 Domain: biomedical Document type: Study Language: en The use of proteinoids for visual identification of the English alphabet, as demonstrated in this study, introduces a new and innovative application of these materials, expanding their capabilities beyond prebiological systems. We have shown that proteinoids possess distinctive features that make them suitable for advanced technological applications, including character recognition and optical computing. Section title: Discussion Educational score: 4.46479606628418 Domain: biomedical Document type: Review Language: en The development of proteinoid-based optical character recognition (OCR) systems represents a significant advancement in biocompatible and environmentally sustainable computing technologies. Proteinoid-based alternatives have unique advantages over conventional electronic OCR systems in biomedical applications that prioritize biocompatibility. For example, these systems have the potential to be integrated into smart contact lenses and implantable medical devices. This would allow for real-time text recognition and information processing within the body, minimizing the risk of negative biological responses. In addition, the biodegradable properties of proteinoids, along with their production from renewable resources, make this technology a more sustainable option compared with conventional electronic OCR systems. This is in line with the increasing global need for environmentally friendly computing solutions. It has the potential to reduce electronic waste caused by current OCR technologies while still being effective. Proteinoids have inherent properties that offer numerous advantages beyond their biocompatibility and sustainability. These structures have the ability to self-assemble, indicating their potential for adaptive learning in demanding environments where conventional electronic systems may not be effective, such as those with extreme temperatures or electromagnetic interference. The adaptability of the technologies of OCR has the potential to greatly increase their operational range. In addition, OCR systems based on proteinoids have the potential to be fundamental components in advanced bioinspired computing structures. This could lead to the development of innovative approaches in chemical intelligence and machine learning that closely mimic the information processing mechanisms found in biological systems. The small size of proteinoid structures allows for the possibility of highly compact OCR systems, which could potentially revolutionize information processing by operating at scales that are currently beyond the reach of conventional electronic systems. This molecular-scale information processing capability has the potential to bring about significant advancements in various fields including nanotechnology and advanced data analytics. It promises remarkable levels of miniaturization and efficiency in tasks such as character recognition and data processing. Section title: Discussion Educational score: 4.467363357543945 Domain: biomedical Document type: Study Language: en We used various methods to analyze our proteinoid material. This ensured reproducibility and confirmed its structure–function relationships. UV experiments showed consistent absorption patterns across batches. 29 FT-IR spectra indicated that peptide bonds formed. 31 We acknowledge that additional analytical methods would provide deeper insights into the material’s molecular architecture. Future studies should use techniques such as circular dichroism (CD) spectroscopy to analyze secondary structures, nuclear magnetic resonance (NMR) spectroscopy to determine sequence distributions, and mass spectrometry to evaluate molecular weight distributions. Adding these characterizations would help create better structure–property relationships. It would also improve the optical recognition of proteinoid-based systems. Our proteinoid-based OCR system works by a unique interplay of charged (Glu/Asp) and aromatic (Phe) residues. This creates many recognition sites through electrostatic and π–π stacking interactions. 15 , 22 The thermal polymerization process creates a mix of peptide sequences. This results in materials with different binding sites and optical properties. 16 , 17 The diverse molecules allow for changes in the optical properties. These depend on specific patterns. This enables character recognition. Proteinoids can self-organize. 21 This allows them to make stable microspheres that reliably react to light. Our SEM analysis supports this. It shows size distributions of 500 nm to 3 μm across various synthesis batches. The use of proteinoids in OCR apps is a big step in bioinspired computing systems. 33 Unlike traditional electronic OCR systems, proteinoid-based recognition uses natural characteristics. It relies on their photoinduced electron transfer mechanisms. 8 , 30 Our studies show that the electrical responses are very specific to certain character patterns. The proteinoid microspheres can, with their unique mechanisms, distinguish between different optical inputs. 25 A deeper look at the molecular mechanisms in this pattern recognition process would improve our understanding of the system’s strengths and weaknesses. Future work should focus on creating structure–activity relationships. Additionally, establishing more rigorous performance metrics for the comparison of proteinoid-based OCR systems with traditional technologies is essential. This work demonstrates the diversity and adaptability of proteinoids, highlighting their capacity to connect prebiological systems with modern technology applications. The effective demonstration of proteinoids in optical character recognition paves the way for further exploration and advancement in the realm of bioinspired materials and computers. Section title: Conclusions Educational score: 4.20745849609375 Domain: biomedical Document type: Study Language: en The use of proteinoids for the optical recognition of the English alphabet signifies a noteworthy progression in the domain of bioinspired materials and computing. This study showcases the multifunctionality and flexibility of proteinoids, highlighting their potential as effective materials for advanced technological applications. We have devised a novel technique for optical character identification by using the distinctive characteristics of proteinoids, such as their capacity to produce stable and selectively permeable microspheres. The findings of this study not only emphasize the effectiveness of proteinoids in carrying out complex tasks such as character identification but also emphasize the importance of exploring the functional abilities of these materials beyond their significance in prebiological phenomena. As research in this subject advances, proteinoids are expected to be used in a wider range of applications including information processing, sensing, and computing. This work provides a basis for future research on the advancement of bioinspired materials and systems. It sets the stage for the development of more sophisticated and effective technologies that rely on the concepts of self-organization and evolutionary continuity. Proteinoids have the potential to provide sustainable, flexible, and intelligent solutions to challenging problems by connecting prebiological systems with modern technological applications. | Review | other | en | 0.999998 |
PMC11696388 | Section title: Introduction Educational score: 3.879314661026001 Domain: other Document type: Study Language: en Wind tunnel tests of aerospace vehicles and aircraft are increasingly making use of pressure-sensitive paint (PSP). PSP is an optical oxygen sensing technique that involves the formulation of an oxygen-sensitive phosphorescent compound, known as a luminophore, and an oxygen-permeable binder into a paint, which is then applied to the surface of a desired test model. 1 When this coated model is placed into a wind tunnel, the static air pressure distribution causes different levels of oxygen diffusion into the binder at different locations. If light of an appropriate wavelength illuminates the model, the luminophore is excited into a singlet excited state and, through intersystem crossing, can be converted to a long-lived triplet excited state (T 1 ). From the T 1 excited state, the luminophore can relax back to the ground state via release of a lower energy photon, through a process called phosphorescence. The phosphorescent intensity is dependent on the local oxygen concentration and therefore, can be imaged and related to the surface pressure through in situ or a priori calibration. 1 The PSP method offers advantages over traditional aerodynamic measurements, such as pressure taps, because of its global resolution and nonintrusive application. Work over the past 30 years has demonstrated the efficacy of PSP in accurately studying various aerodynamic applications such as steady and unsteady flows, 2 − 9 rotating flows, 10 − 12 cryogenic experiments, 13 − 15 and even blast waves. 16 , 17 Section title: Introduction Educational score: 4.106067657470703 Domain: biomedical Document type: Study Language: en One of the main drawbacks concerning PSP measurements is the inherent temperature sensitivity, resulting in inaccurate pressure determination. This results from nonradiative decay of the luminophore T 1 state and temperature-dependent oxygen diffusion through the paint binder. 1 Attempts to resolve the temperature sensitivity issue have involved the development of dual-luminophore PSPs that contain a pressure-sensitive luminophore and a secondary temperature-sensitive but pressure-insensitive luminophore. The secondary signal can then be used to account for errors in temperature change associated with the primary luminophore signal. 5 , 18 , 19 However, some dual-luminophore formulations suffer from luminophore cross-talk which can hinder PSP performance. 20 , 21 Section title: Introduction Educational score: 4.388214111328125 Domain: biomedical Document type: Study Language: en PSP development in recent years has largely focused on binder development, such as new polymers, 22 − 26 poly ceramic PSP (PC-PSP), 27 , 28 and anodized aluminum PSP (AA-PSP), 29 − 31 in an attempt to improve response times for unsteady flow measurements. However, a distinct lack of development of the luminophore species has been pursued. Luminophore molecular design can be used to tailor the lifetime/quantum yield of emission and the molar absorptivity of the resulting PSP, thus affecting properties like pressure sensitivity S p , temperature sensitivity, S T , and the brightness of emission. Additionally, the solubility of a luminophore, and its interaction with the binder matrix, can greatly impact an optical molecular-based sensor’s performance. 32 Traditionally, PSP formulations have used Pt(II) porphyrins, Ru(II) polypyridyls, and pyrene derivatives as the luminophore. 1 Ru(II) polypyridyls have high quantum yields and adsorb well onto porous binders. 33 However, Pt(II) porphyrin-based PSPs typically have a higher S p and a relatively lower S T but do not adsorb well onto porous binders. For traditional polymer-based PSPs, the luminophore Pt(II)-5,10,15,20-tetrakis(2,3,4,5,6-pentafluorphenyl)-porphyrin (PtTFPP) is most ubiquitously used due to its high degree of photostability and solubility in many common PSP polymers. 34 Indeed, the only commercially available PSPs utilize PtTFPP as the luminophore. 35 We recently explored the effect of tetraphenyl porphyrin degree, type, and position of phenyl halogenation on PSP performance and found these factors can have a large effect on the subsequent polymer-based PSP performance. 36 This was especially true with S T , finding that PtTFPP polystyrene PSPs possessed the highest S T . Through luminophore design, we demonstrated that S T can be reduced by 0.5%/K. However, the S p in comparison was largely unaffected by porphyrin phenyl halogenation due to their similar lifetimes in the polymer binder matrix. Considering these results, this study investigates the effect of the central metal ion on the photophysics of freebase and metal complexes of TFPP and the resulting effect on polymer-based PSP performance. Typical phosphorescent metalloporphyrins constitute Pt(II), Pd(II), and Ir(III). Therefore, we introduce Ir(III)(Cl)(CO)TFPP ( Ir1 ) for the first time in PSP formulations along with PtTFPP ( Pt1 ) and PdTFPP ( Pd1 ) to explore the effect of the central metal ion on the PSP performance . Additionally, Zn(II)TFPP ( Zn1 ) and the freebase porphyrin, TFPP ( Fb1 ), were included to study fluorescent porphyrins in polymer-based PSPs. Section title: Experimental Section Educational score: 1.6821894645690918 Domain: biomedical Document type: Other Language: en Detailed synthetic procedures and characterization data can be found in the accompanying Supporting Information . Section title: Photophysical Studies Educational score: 4.290398597717285 Domain: biomedical Document type: Study Language: en UV–vis electronic absorption spectra were recorded on a Mettler Toledo UV5Bio spectrophotometer. Steady-state emission and excitation spectra and lifetime data were recorded on an Edinburgh Instruments FP920 Phosphorescence Lifetime Spectrometer equipped with a 450 W steady state xenon lamp, a 5 W microsecond pulsed xenon flash lamp , interchangeable EPL pulsed diode lasers, and a red-sensitive photomultiplier in Peltier (air cooled) 53 housing (Hamamatsu R928P). Plotting, fitting, and analysis of data were carried out using Origin 2019b. All data were fitted with exponential decay models and the goodness of fit evaluated by residual, χ 2 , and R 2 analysis. These measurements were recorded in chloroform which was dried over 4 Å molecular sieves and degassed using three freeze–pump–thaw cycles and standard Schlenk techniques. All samples were prepared in an Innovative Technologies System Two, under argon, where the concentration of oxygen and water was always kept below 0.1 ppm. Emission spectra and lifetime measurements were recorded in J. Young’s taps sealed quartz cuvettes. Quantum yields were calculated using the relative method, with tetraphenyl porphyrin in toluene (Φ = 0.07) as a standard. Polystyrene-doped samples were prepared by drop casting approximately 100 μL of the PSP polystyrene solution in chloroform onto a glass microscope slide and air-dried. These samples were subsequently taken into an argon-filled glovebox to remove all residual volatiles and sealed with another glass slide using vacuum grease around the edges to prevent diffusion of oxygen into the sample. The reported data are an average of three independent measurements. The weight-averaged emission lifetimes in argon-saturated polystyrene, ⟨τ⟩ were calculated using eq 1 . 1 where B is the initial intensity of a given component and τ is the lifetime of a given component. Section title: PSP Recipes Educational score: 3.8450112342834473 Domain: biomedical Document type: Study Language: en The polystyrene-based PSPs were formulated by dissolving 1 g of polystyrene (Sigma-Aldrich M w ∼ 380,000) in 25 mL of chloroform and using a luminophore loading of 0.68% wt/wt luminophore/polystyrene. Section title: PSP Performance Studies Educational score: 2.5861847400665283 Domain: biomedical Document type: Study Language: en PSP formulations were sprayed onto Ambersil matt white RAL 9010 base-coated aluminum coupons using a spray gun in 12 light coats. The samples were left to air-dry for 30 minutes after spraying. Section title: PSP Performance Studies Educational score: 2.3542277812957764 Domain: biomedical Document type: Study Language: en The performance of the PSP formulations was investigated in the standard approach of a priori calibration using the University of Manchester PSP calibration chamber. The a priori calibration was performed using the published procedure. 36 Section title: PSP Performance Studies Educational score: 4.057438850402832 Domain: biomedical Document type: Study Language: en The pressure sensitivity at a certain temperature, S P ( T ) was calculated from the slope of the modified Stern–Volmer calibration plots using 2 with I ref and P ref as the luminescence intensity and pressure, respectively, at 100 kPa and 293 K. 2 Section title: PSP Performance Studies Educational score: 3.80184006690979 Domain: biomedical Document type: Study Language: en The temperature sensitivity at a given pressure, S T ( P ), is calculated as the percentage change in I ref / I with respect to the temperature by using 2 with I ref as the luminescent intensity at 100 kPa and 293 K. 3 Section title: PSP Performance Studies Educational score: 4.018553733825684 Domain: biomedical Document type: Study Language: en Photodegradation studies were conducted at room temperature and pressure and involved recording images of the PSP luminescence every 3 min for 45 min of constant illumination. The amount of photodegradation was calculated as the % luminescent intensity (at 45 min) of the original luminescent intensity (at 0 min). Section title: Synthesis Educational score: 4.151970863342285 Domain: biomedical Document type: Study Language: en The freebase porphyrin Fb1 was synthesized via the standard Lindsey conditions in a 19% yield. 37 The Zn(II) complex ( Zn1 ) was synthesized by heating Fb1 with Zn(OAc) 2 to reflux in a chloroform–methanol mixture, giving an isolated yield of 86%. The Ir(III) complex ( Ir1 ) was synthesized by heating Fb1 with Ir(COD) 2 Cl 2 to reflux in 1,2,4-trichlorobenzne, affording an isolated yield of 46%. 38 The Pd(II) and Pt(II) ( Pd1 and Pt1 ) complexes were synthesized by heating PdCl 2 /PtCl 2 to reflux in benzonitrile under argon, before adding Fb1 , resulting in isolated yields of 92% and 87%, respectively (see the accompanying Supporting Information for full synthetic procedure and characterization data). 36 Section title: Single-Crystal X-ray Crystallography Educational score: 4.406861305236816 Domain: biomedical Document type: Study Language: en Suitable crystals for single-crystal XRD were grown through slow evaporation of a 1:1 DCM/hexane mixture for Zn1 , Pd1 and 1:1 DCM/chloroform for Ir1 . The single-crystal XRD structure for Pt1 has been previously published . 36 Normal coordinate structural decomposition (NSD) was used to evaluate the in-plane (IP) and out-of-plane (OOP) distortions adopted by the different porphyrins in their single-crystal geometries. NSD analysis was performed using the web-based program developed by Kingsbury and Senge. 39 The structures of the metalloporphyrins are all relatively flat, with Pt1 , Pd1 , and Ir1 all having a small Δoop = 0.03 Å and the metal ion sitting in the plane of the 4 nitrogen atoms ( Table 1 ). Pt1 and Pd1 adopt a slight wave ( E gx / E gy ), while Ir1 adopts a slight dome (A 2u ) conformational distortion. Zn1 has a much larger Δoop = 0.15 Å than the other metalloporphyrins, adopting a wave ( E gx / E gy ) conformational distortion. The porphyrins also possess IP distortions, which are expansions of the porphyrin core around the central metal ion. Zn1 has the highest Δip = 0.19 Å and a Zn–N bond distance of 2.044(14) Å, which is consistent with other Zn(II) porphyrins, Zn–N(range) = 2.298(7) – 1.799(8) Å. 40 Zn(II) has low-energy 3d orbitals, which are unable to π-backbond with the high-energy porphyrin π* orbitals; therefore, a weak and long Zn–N bond is formed. Looking at Pd1 , the Δip = 0.08 Å and the Pd–N bond distance = 2.017(16) Å, which is consistent with other Pd(II) porphyrins, Pd–N(range) = 2.131(4) – 1.926(12) Å. 40 The Pd(II) ion forms a stronger M–N interaction because of the higher in energy 4d orbitals, and thus greater π-backbonding can occur. Pt1 has a Δip = 0.07 Å and a Pt–N bond distance of 2.009(2) Å, which is consistent with other Pt(II) porphyrins, Pt–N(range) = 2.069(4) – 1.960(10) Å. 40 For the Pt(II) porphyrin, the M–N interaction is even stronger because the Pt(II) ion has even higher energy 5d orbitals, which facilitates even stronger π-backbonding. The Ir–N bond in Ir1 is markedly longer at 2.050(3) Å. Section title: UV–Vis Electronic Absorption Spectroscopy Educational score: 4.046516418457031 Domain: biomedical Document type: Study Language: en The UV–vis electronic absorption spectra of all metalloporphyrins in chloroform were found to be characteristic of typical metalloporphyrins, with an intense Soret band (B band) around 400 nm and two much weaker in intensity Q bands centered on 550 nm . Section title: UV–Vis Electronic Absorption Spectroscopy Educational score: 4.486265182495117 Domain: biomedical Document type: Study Language: en The spectral features can be explained using Gouterman’s four orbital model, where the four Frontier orbitals: highest occupied molecular orbital (HOMO)–1 (a 1u ), HOMO (a 2u ), and two degenerate lowest unoccupied molecular orbitals (LUMOs) (e g ) are relatively isolated in energy. 41 The Soret band is formed predominantly of a π–π* transition from HOMO–1 to the degenerate LUMOs. Whereas the Q bands are formed of Q abs (1,0) and Q abs (0,0), which are assigned as a vibronic satellite and the electronic origin π–π* transitions from the HOMO to the degenerate LUMOs. Freebase porphyrins (Fb) are known to have a further split in each Q-band due to the symmetry break of the central proton axis. Consequently, for Fb1 , there are two sets of Q bands, Q y and Q x , along with their accompanying vibrational satellites, resulting in four Q bands in total. The position of the Soret band blue-shifts in the order of Ir1 = 414 nm and Zn1 = 414 nm > Fb1 = 412 nm > Pd1 = 407 nm > Pt1 = 392 nm. The reason for this blueshift has recently been revisited with Ghosh et al. finding that the metal ions stabilize the a 2u HOMO–1 increasing the HOMO–1 to LUMOs energy gap and thus the Soret band energy. 42 The intensity of the Q abs (0,0) band increases across the family of porphyrins. In contrast, the intensity of the vibronic satellite, Q abs (1,0), remains roughly consistent because it derives its absorption strength from the B transitions (that make up the Soret band) via Herzberg–Teller coupling, which exceeds the intensity from the normal Franck–Condon progressions. 43 The increasing intensity of Q abs (0,0) can best be seen using the ratio of the integrated intensity of Q abs (0,0) to Q abs (1,0) band (because the intensity of the Q(1,0) band remains roughly constant), which increases in the order of Fb1 = 0.12 < Zn1 = 0.25 < Ir1 = 0.34 < Pd1 = 0.53 < Pt1 = 0.79. For the heavily fluorinated porphyrin (TFPP) used in this study, the a 2u MO is already greatly stabilized and so the freebase porphyrin, Fb1 , has its a 2u MO and a 1u MO flipped. Therefore, the a 2u becomes the HOMO–1 and the a 1u the HOMO. 44 As metal ions are incorporated into this porphyrin, the a 2u HOMO–1 will be increasingly stabilized, with the strength of this stabilization dependent on the electronegativity of the metal. Therefore, any metal ion will stabilize the a 2u HOMO–1 away from the a 1u HOMO, decreasing orbital mixing and increasing Q abs (0,0) intensity. The exception to this trend is Ir1 , which has a reduced intensity Q abs (0,0) band when compared to Pd1 , despite Pd(II) and Ir(III) having similar electronegativity values (2.70 and 2.79, respectively). 45 The axial Cl and CO act as electron-withdrawing substituents, which can lower the energy of the a 1u HOMO, thus bringing it closer to the a 2u HOMO–1, therefore, increasing the level of orbital mixing, which in turn reduces Q abs (0,0) intensity. Section title: Density Functional Theory and Time-Dependent-Density Functional
Theory Calculations Educational score: 4.1685638427734375 Domain: biomedical Document type: Study Language: en To further explore the electronic structure of Fb1 , Zn1 , Ir1 , Pd1 , and Pt1 , DFT and time-dependent DFT (TD-DFT) calculations were carried out. Calculations employed either the B3LYP 46 (DFT) or CAM-B3LYP 47 (TD-DFT) functional with the lanl2dz 48 basis set for all atoms. Solvation in chloroform was treated with a conductor-like polarizable continuum model and the D4 charge-dependent atom-pairwise dispersion correction 49 was included throughout. This methodology has been found to be optimal in previous porphyrin DFT and TD-DFT calculations. 50 All structures are optimized and confirmed as minima on the potential energy surface, through the absence of imaginary vibrational modes. All calculations were performed by using the Orca 5.0.4 software package. Section title: DFT Calculations Educational score: 4.18541955947876 Domain: biomedical Document type: Study Language: en All calculated structures are near-flat. The relative energies of HOMO–1, HOMO, LUMO, and LUMO+1, with the accompanying relevant energy gaps, are found in Figure 4 and Table S1 . The porphyrins possess an energetically isolated degenerate LUMO and LUMO+1 (except Fb1, which has the LUMOs split in energy by 0.01 eV, due to the central proton axis breaking symmetry) and a close in energy HOMO and HOMO–1. The HOMO and HOMO–1 are a 2u and a 1u π MO and are identical across all of the systems studied. Section title: DFT Calculations Educational score: 4.475555419921875 Domain: biomedical Document type: Study Language: en Comparing Fb1 to Zn1 , the incorporation of the Zn(II) ion raises the HOMO and HOMO–1 in energy, and they also become closer in energy, with HOMO to HOMO–1 gaps of 0.11 and 0.03 eV, respectively. For Pt1 , Pd1 , and Ir1 , the DFT predicts that the a 2u MO is increasingly stabilized by the increasingly electronegative metal ions, thus causing the a 2u MO to become the HOMO–1 as it is stabilized below the a 1u MO in Pd1 and Pt1 . Therefore, the HOMO to HOMO–1 energy gap increases in the order of Ir1 = 0.01 eV < Pd1 = 0.08 eV < Pt1 = 0.13 eV. The a 2u MO of Pt1 and Ir1 are predicted to be at similar energies (−6.40 and −6.41 eV, respectively), owing to the relatively similar electronegativities of the metal ions (2.98 and 2.79, respectively). However, for Ir1 the a 1u MO is greatly stabilized and the a 2u MO is slightly stabilized. This makes the two MOs almost degenerate in energy, contributing to the much smaller HOMO to HOMO–1 energy gap for Ir1 . The average of the HOMO–1 to LUMO and HOMO to LUMO energy gaps affords the center of gravity of the resulting absorption spectrum, which then determines Soret band energies. The center of gravity of the absorption spectrum is predicted to increase steadily in the order of Fb1 = 2.89 eV < Zn1 = 2.93 eV < Ir1 = 3.04 eV < Pd1 = 3.08 eV < Pt1 = 3.16 eV because the a 2u MO is being stabilized away from the a 1u MO. The exception for this is Ir1 , which has a similar a 2u MO energy to Pt1 , but its degenerate LUMOs are predicted to be stabilized by about 0.2 eV with respect to the other metalloporphyrins, leading to smaller HOMOs to LUMOs energy gaps. Section title: TD-DFT Calculations Educational score: 4.503978729248047 Domain: biomedical Document type: Study Language: en TD-DFT calculations predict the UV–vis electronic spectra generally well, with each porphyrin having four major excited states, except for Pd1 ( Table S3 ), in line with the four-orbital model. The absorption spectra of the metalloporphyrins are predicted to have Soret and Q bands, each made of two symmetry matched excited states, B y /B x and Q y /Q x , respectively (with B y and B x forming the Soret band and Q y and Q x forming the Q abs (0,0) feature). The freebase porphyrin, Fb1 , is predicted to have two symmetry split excited states for each absorption feature due to the central proton axis energetically splitting the LUMO and LUMO+1. Surprisingly, the Soret band feature of Pd1 is predicted to be formed of three degenerate excited states. Across the series, Soret bands are predicted to blue-shift in wavelength, which matches the experimental data. This blueshift is due to the increasing energy gap between the LUMOs and the center of gravity of the absorption spectrum, with increasing metal ion electronegativity. However, the exception is Ir1 with its lower in energy LUMOs, which decrease the HOMOs to LUMOs energy gap. Therefore, Ir1 possesses a red-shifted Soret band, with respect to Pd1 and Pt1 . The oscillator strength of the Q-band feature is predicted to increase steadily from Fb1 ≈ 0 < Zn1 = 0.001 < Ir1 = 0.003 < Pd1 = 0.010 < Pt9 = 0.021. This trend aligns with the experimental data, where the intensity of the Q abs (0,0) band increased across the series. The four orbital model predicts that each excited state is made up of a pair of one-electron transitions from the HOMO–1 and HOMO to the LUMOs. 41 Different combinations of these one-electron transitions can happen constructively, leading to the intense Soret band feature, or destructively, leading to the much weaker Q-band features. 51 The more mixing of the electron transitions, the more destructive combinations are possible and thus the weaker the Q bands. This orbital mixing is dependent on the energy gap between the HOMO and HOMO–1 orbitals in the ground state, which is most evident when comparing the sum of the predicted contributions of the transitions from the HOMO–1 to the LUMOs and the HOMO to the LUMOs, for a given excited state. Focusing on the Q x excited state, for example, , the sum of the contributions of the HOMO to LUMOs transitions become increasingly more dominant over the sum of the contributions of the HOMO–1 to LUMOs transitions in the Q x excited-state makeup across the series. Section title: TD-DFT Calculations Educational score: 4.402693748474121 Domain: biomedical Document type: Study Language: en The increasing dominance of the HOMO to LUMOs transitions (and therefore, decreasing dominance of HOMO–1 to LUMOs transitions) in the Q x excited-state makeup, follows the progression Fb1 = 53% ≈ Zn1 = 52% ≈ Ir1 = 53% < Pd1 = 59% < Pt1 = 62%. The HOMO to LUMOs transitions become increasingly dominant in the excited-state composition from Zn1 to Pd1 to Pt1 due to the increasing stabilization of the a 2u MO, resulting in a greater HOMO to HOMO–1 energy gap. However, for Ir1 the a 1u MO is also stabilized, which decreases the HOMO to HOMO–1 energy gap. As less orbital mixing occurs in the excited state, the Q bands increase in intensity because fewer destructive combinations are possible. It is useful to look at the resulting MO energy levels in the excited structures to better explain the excited-state composition ( Table S2 ). Upon excitation, the now singly occupied molecular orbital (SOMO)–LUMO/LUMO+1 destabilize in energy and the now SOMO–HOMO/HOMO–1 stabilize in energy. The a 2u MO stabilizes in energy to a greater extent than the a 1u MO, causing the a 2u MO to become the HOMO–1 in all metalloporphyrins. Whereas in Fb1 , the a 1u MO remains as the HOMO–1. Consequently, the HOMO to HOMO–1 energy gaps in the excited structures are Fb1 = 0.01 eV < Zn1 = 0.06 eV < Ir1 = 0.08 eV < Pd1 = 0.16 eV < Pt9 = 0.22 eV, which matches the experimentally observed increasing intensity of the Q abs (0,0) feature across this series. Section title: Emission Spectroscopy Educational score: 4.323888778686523 Domain: biomedical Document type: Study Language: en The emission spectra of the porphyrins in chloroform possess 2 distinct bands, the electronic origin of the emission, denoted as Q em (0,0) and a lower in energy vibronic satellite, denoted as Q em (0,1) . The Q em (0,0) maxima follow the same trend as the Soret maxima with Ir1 = 680 nm > Pd1 = 672 nm > Pt1 = 651 nm. However, the Q em (0,0) maximum of Zn1 is blue-shifted, with respect to the other metalloporphyrins to 599 nm, which is common for Zn(II) porphyrins. 52 Similar to the absorption spectra, the Q em (0,0) feature increases in intensity with increasing metal ion electronegativity, for example, the integrated intensity of Q em (0,0)/Q em (0,1), increases in the order of Fb1 = 0.26 > Zn1 = 0.16 < Ir1 = 0.98 < Pd1 = 1.30 < Pt1 = 1.76. The increasing intensity of the spectral features suggests that the HOMO to HOMO–1 energy gap is increasing across this series. Section title: Emission Spectroscopy Educational score: 4.308834552764893 Domain: biomedical Document type: Study Language: en Lifetimes, τ (Ar) and quantum yields, Φ (Ar) , were measured in argon-saturated solutions and can be split into two categories: fluorescent and phosphorescent porphyrins. Focusing on the fluorescent porphyrins, Fb1 and Zn1 , their lifetimes are much shorter, 9.6 and 1.3 ns, respectively, which is typical of fast fluorescence from the S 1 state. Incorporation of the Zn(II) ion promotes intersystem crossing from the S 1 state to the T 1 state through increased spin orbital coupling via the heavy atom effect. Therefore, Zn1 has a much shorter τ (Ar) and a lower Φ (Ar) . Moving onto the phosphorescent porphyrins, their emission lifetimes are much longer, on the order of μs, indicating slow phosphorescence from the T 1 state. The phosphorescence τ (Ar) and Φ (Ar) of Pt1 (τ (Ar) = 49.6 μs and Φ (Ar) = 0.082) and Pd1 (τ (Ar) = 748.2 μs and Φ (Ar) = 0.039) are governed mainly by the increasing heavy atom effect of the Pt(II) compared with that of the Pd(II) ion, which decreases τ (Ar) and increases Φ (Ar) . Ir1 by comparison has a smaller Φ (Ar) = 0.022 than both Pt1 and Pd1 and a τ (Ar) = 90.0 μs, which is longer than Pt1 but shorter than Pd1 . Section title: Polystyrene PSP Performance Studies Educational score: 4.092403411865234 Domain: biomedical Document type: Study Language: en The metalloporphyrins were formulated into polymer PSPs, using the polystyrene formulation from our previous luminophore comparison study. 36 In this study, however, a higher M w of polystyrene was used ( M w = 380,000). Polystyrene is not the optimal polymer for PSP formulations but was deemed suitable for luminophore comparison studies. The performance metrics studied are pressure sensitivity, S p , temperature sensitivity, S T , and photodegradation after constant illumination at room temperature and pressure for 45 min. Section title: Pressure Sensitivity, S p Educational score: 4.374731063842773 Domain: biomedical Document type: Study Language: en Pressure sensitivity, S p , is a crucial performance metric for PSPs; a higher sensitivity to pressure allows for smaller pressure changes to be resolved. Stern–Volmer calibrated responses at 273, 293, and 313 K are presented below . S p is calculated as the change in I ref / I , with respect to P / P ref , and is quoted at 293 K (denoted as S p (293 K)). The phosphorescence lifetime, τ (Ar) , of the luminophore is an important determining factor of S p ; the longer the T 1 excited state exists, the more chances there are for collisional quenching with diffused O 2 . There is no significant change in the emission and excitation spectra of the porphyrins in polystyrene. The phosphorescent lifetimes in argon-saturated polystyrene were found to be biexponential for the phosphorescent porphyrins (this is common for luminophores immobilized in a polymer matrix) and monoexponential for the fluorescent porphyrins. 53 For the phosphorescent porphyrins, the S p (293 K) increases in the order of Pt1 = 0.662 < Ir1 = 0.824 < Pd1 = 0.913, which correlates to the trend in the τ (Ar) of these luminophores in polystyrene. Therefore, Ir1- and Pd1- containing PSPs are much more sensitive to pressure than Pt1 -containing PSPs and are attractive options for improved PSP formulations. The small changes in I ref / I with increasing pressure for Fb1 and Zn1 , make accurate performance determination unreliable, and they would not be able to accurately sense large changes in pressure. The fluorescent porphyrins had S p (293 K) = 0.019 for Fb1 and 0.007 for Zn1 . On the fluorescent time scale of nanoseconds these luminophores are very limited by oxygen diffusion through the polystyrene binder. However, the reduced fluorescent lifetime of Zn1 may make it an attractive option for fast responding porous PSPs, where sensing is theoretically not limited by oxygen diffusion through the binder and thus is something we plan to explore in the future. 54 Overall, the S p (293 K) of the phosphorescent porphyrin PSPs presented here varies by 38%, highlighting that the nature of the central metal ion can have a substantial effect on S p . Section title: Temperature Sensitivity, S T Educational score: 4.390942573547363 Domain: biomedical Document type: Study Language: en The temperature sensitivity, S T , of PSPs is also an important performance characteristic. PSPs and polymer PSPs especially possess a high inherent temperature sensitivity, which makes reliable pressure determination more convoluted. The temperature sensitivity of PSPs is due to two factors: at higher pressures the temperature-dependent oxygen diffusion through the binder dominates and at lower pressures the intrinsic temperature dependence of the nonradiative deactivation of the luminophore excited state dominates. 1 A low S T , single luminophore PSP would be ideal to avoid the complexity associated with binary PSP formulations. The long-lived T 1 excited state has a temperature dependency due to vibrational, rotational, and collisional quenching. Keeping the polymer binder the same, while changing the luminophore, theoretically allows investigation of the luminophore effect on PSP temperature sensitivity. S T is calculated as the change in I ref / I with respect to the temperature and is typically quoted at 100 kPa, denoted as S T (100 kPa). Looking at the phosphorescent porphyrins first, it can be seen that the S T (100 kPa) increases in the following order: Pt1 = 1.56%/K < Ir1 = 1.62%/K < Pd1 = 1.71%/K, aligning well with their increasing emission lifetimes. The change in S T with increasing pressures is also presented for Ir1 , Pd1 , and Pt1 . For Ir1 and Pd1 , S T (10 kPa) ≈ 0.20%/K; for Pt1 , it is slightly higher, with S T (10 kPa) = 0.32%/K. As the pressure increases, S T increases linearly for Ir1 and Pt1 . However, the increase is greater for Ir1 than for Pt1 , causing the S T of Ir1 to become greater than that of Pt1 at roughly 65 kPa. At higher pressures, there is more oxygen in the binder matrix, and the longer lived T 1 lifetime of Ir1 , compared to Pt1 , will facilitate more deactivation by this increased O 2 concentration. Therefore, Ir1 at these higher pressures will be more susceptible to the increasing collisions with O 2 because of the increasing energy of diffused O 2 , which accompanies increasing temperatures. Pd1 has an even longer T 1 lifetime than Ir1 and so its S T becomes greater than that of Pt1 earlier (roughly 40 kPa). However, in contrast to Ir1 and Pt1 , for Pd1 , a second-order polynomial fit was found to better fit the change in S T with pressure, compared to a linear fit ( R 2 = 0.999 for second-order polynomial and 0.991 for linear). This nonlinear behavior is probably due to excess quenching of Pd1 at higher pressures and consequently causes the S T of Pd1 to plateau and subsequently dip below that of Ir1 at pressures >130 kPa. These findings are interesting, as they implicate that the more pressure-sensitive Ir1 and Pd1 PSPs could be used in low-pressure (<70 kPa) measurements where their S T is lower and their low quantum yields (at high pressures) are less of an issue. On the other hand, Pt1 -based PSPs are more suited to high pressure (>70 kPa) applications, where the S T is lower (compared to Ir1 and Pt1 ) and the higher quantum yields are more desirable. The two fluorescent porphyrins, Fb1 and Zn1 , exhibit weak temperature sensitivities, 0.13 and 0.11%/K, respectively, due to their much shorter fluorescent lifetimes. Section title: Photodegradation Educational score: 4.186605453491211 Domain: biomedical Document type: Study Language: en Photodegradation of the phosphorescent porphyrins Ir1 , Pd1 , and Pt1 polystyrene PSPs was also investigated. The photodegradation is calculated as the % luminescent intensity of the original luminescent intensity (at 0 min) after 45 min of constant illumination at room temperature and pressure. A high-performing PSP formulation will have minimal photodegradation during testing to facilitate greater usage over multiple tests. Photodegradation of PSPs during testing occurs primarily via the formed singlet oxygen radical, which can go on to damage the luminophore and binder, leading to a reduction in paint performance. 55 Pt1 photodegrades the most after 45 min, with a 6.6% reduction in luminescence intensity . Pd1 is the most photostable, with a 4% reduction in luminescence intensity after 45 min of constant illumination. Ir1 is more photostable than Pt1 , with a 5.5% photodegradation after 45 min. However, the photodegradation of Ir1 is noticeably more linear in this time scale (compared to Pt1 ), which begins to tail off toward the end of the measurement. Section title: Conclusions Educational score: 4.404227256774902 Domain: biomedical Document type: Study Language: en We have investigated a family of metalloporphyrins and their parent free-base ligand TFPP to examine the effect of the central metal ion of porphyrins on the electronic structure, photophysics, and the resulting polymer-based PSP performance. The Pt(II) complex, PtTFPP ( Pt1 in this study), is used almost ubiquitously in polymer-based PSP formulations and therefore we presented Pd(II) ( Pd1 ), Zn(II) ( Zn1 ), and, for the first time in the PSP field, Ir(III) ( Ir1 ) TFPP complexes. The central metal ion mainly affects the energy of the occupied a 2u MO through metal electronegativity-dependent stabilization. Preferential stabilization of the a 2u MO by more electronegative metals widened the HOMOs to LUMOs energy gap, resulting in a blueshift of the spectra. However, Ir1 was an exception, as it was found using DFT calculations to have stabilized LUMOs relative to the other metalloporphyrins, resulting in red-shifted spectral features. The preferential stabilization of the a 2u away from the a 1u MO increases the HOMO to HOMO–1 energy gap and thus increases the intensity of Q(0,0) feature in the absorption and emission spectra. Ir1 was found to have a greatly stabilized a 1u MO, brining it closer in energy to the a 2u MO, thus reducing the intensity of the Q(0,0) features. TD-DFT calculations showed that the intensity of the Q abs (0,0) feature increased due to the contributions of the electronic transitions from the HOMO–1 and the HOMO to the LUMOs, in the excited-state makeup decreasing and increasing, respectively, with increasing HOMO–1 to HOMO energy gaps. The heavy metal effect of the Zn(II) ion caused Zn1 to have a reduced fluorescence lifetime and quantum yield, compared to the freebase porphyrin Fb1 . On the other hand, Pd1 , Ir1 , and Pt1 exhibited room-temperature phosphorescence, with Pt(II) having a larger quantum yield (0.039 and 0.082, respectively) but shorter lifetime of emission (748.2 and 49.6 μs, respectively) due to the increased heavy atom effect of Pt(II) over Pd(II). Ir1 had a smaller quantum yield (0.022) than Pd1 but a longer lifetime of emission (90.0 μs) compared to that of Pt1 . The phosphorescent porphyrins Ir1 , Pd1 , and Pt1 , were all viable candidates as luminophores in PSP formulations. The pressure sensitivity at 293 K, S p (293 K), of the resulting polystyrene PSPs greatly increased in the order of Pt1 = 0.662 < Ir1 = 0.824 < Pd1 = 0.913, in line with the increasing phosphorescence lifetimes. Increasing the phosphorescence lifetime of the luminophore was found to increase the temperature sensitivity ( S T ) at higher pressures, with Ir1 and Pd1 having higher S T at higher pressures than Pt1 . However, at lower pressures, Pt1 had the highest S T and Ir1 / Pd1 had a lower S T . Therefore, the nature of the central metal ion in porphyrin luminophores can be used to tailor PSP formulations for specific pressure measurement. The quantum yields of emission for Pd1 and Ir1 are also much lower than for Pt1 , reducing the brightness of these PSPs. Finally, this work shows that the central metal ion of porphyrin luminophores can be used to tune the pressure and temperature sensitivity of the resulting PSP formulation for the desired application. This work contrasts our previous study on the effect of porphyrin phenyl halogenation, which mainly affected the resulting temperature sensitivity of the PSPs. 36 We hope this research can assist with making new and improved PSP formulations. | Study | other | en | 0.999999 |
PMC11696396 | Section title: Introduction Educational score: 3.9725587368011475 Domain: biomedical Document type: Review Language: en Dendrimers are highly branched, three-dimensional polymeric macromolecules. Their tunable architecture allows for precise control over size, shape, and surface functionality. 1 , 2 Due to these characteristics, dendrimers are utilized in various biomedical applications, including drug delivery and imaging. 3 − 5 Low-generation dendrimers, with molecular weights up to ∼30,000 Da and sizes of up to ∼6 nm, are employed in the majority of preclinical biomedical investigations. 3 , 4 , 6 − 9 Section title: Introduction Educational score: 3.7302024364471436 Domain: biomedical Document type: Review Language: en Recently, dendrimers have advanced into human clinical trials. 10 − 14 Promising applications include the use of drug-conjugated PEGylated polylysine dendrimers for cancer treatment. 12 In addition, G4-OH dendrimers conjugated to N-acetyl cysteine, an anti-inflammatory and antioxidant agent, have been assessed for treating hospitalized patients with severe COVID-19. 11 , 13 Section title: Introduction Educational score: 3.765594720840454 Domain: biomedical Document type: Study Language: en As dendrimers become more prevalent in clinical trials, a rigorous preclinical safety evaluation is essential to identify potential toxicity concerns prior to patient administration. 15 − 20 In particular, unintended interactions between dendrimers and blood components could disrupt the delicate hemostatic balance and lead to life-threatening complications. Section title: Introduction Educational score: 4.684045791625977 Domain: biomedical Document type: Study Language: en In normal blood clotting, the exposure of tissue factor (TF) within the lumen of injured blood vessels triggers the coagulation cascade that ultimately leads to the thrombin-mediated proteolytic cleavage of fibrinogen into insoluble fibrin . 21 The resultant fibrin network, in conjunction with blood cells, forms the thrombus (or blood clot) that seals the injured blood vessel and stops bleeding. Subsequently, the fibrinolytic system acts over time to dissolve the formed thrombus and allow the restoration of normal blood flow. Blood clotting can also be triggered by contact with exogenous anionic surfaces, such as nanomaterials or biomaterials. 22 − 24 In this scenario, the coagulation cascade is initiated by activating the plasma contact system, a process that involves conversion of the factor XII (FXII) zymogen into the proteolytically active form FXIIa . 21 , 25 We further emphasize that the formation of fibrin clots having proper structural and biomechanical properties—including fiber thickness, network density/porosity, permeability, and stiffness/plasticity—is crucial for ensuring effective hemostasis and wound healing. 26 − 28 Section title: Introduction Educational score: 4.331238746643066 Domain: biomedical Document type: Study Language: en Previous studies have explored the interactions and potential effects of dendrimers on blood cells, 29 − 33 as well as their impact on the contact, coagulation, and fibrinolytic systems. 34 − 39 It has been found that dendrimers can modulate the activity of coagulation enzymes and cause alterations to key hemostatic parameters such as thrombin generation and clotting time, depending on dendrimer generation, concentration, and surface chemistry. However, there has been limited attention given to the integrated impact of dendrimers on the formation, structure, properties, and stability of fibrin clots. Addressing this research gap is crucial, since abnormal clot formation can pose a significant risk for individuals. 26 , 40 − 42 Indeed, various diseases and pathologies can result in altered clotting kinetics and impaired clot structure, leading to an increased tendency for bleeding or thrombosis. For example, hematologic conditions like hemophilia A and FXI deficiency increase susceptibility to bleeding, 41 , 43 whereas individuals with cardiovascular disease, type-2 diabetes, and cirrhosis face an elevated risk of thrombosis. 42 , 44 − 47 Additionally, a recent study explored the clot formation process on blood-contacting medical devices, revealing the influence of material wettability on fibrin network structure and stability, and the potential implications for material-induced thrombosis. 48 Section title: Introduction Educational score: 4.134701251983643 Domain: biomedical Document type: Study Language: en We have recently explored how anionic ultrasmall gold nanoparticles (usGNPs) with a hydrodynamic diameter of about 3.5 nm—similar to polyamidoamine (PAMAM) G3/G4 generation dendrimers—interfere with the coagulation system and impact fibrin clot formation. 49 − 51 These ultrasmall particles interacted with fibrinogen, delayed the kinetics of clot formation, and disrupted the normal architecture of the fibrin network, leading to larger clot pore sizes and increased clot permeability to liquid. These previous findings imply the potential for similar adverse effects of dendrimers on the fibrin clot formation process. Section title: Introduction Educational score: 4.085456371307373 Domain: biomedical Document type: Study Language: en Based on the above considerations, herein we employ an array of analytical techniques to explore the impact of dendrimers on the formation, structure, properties, and stability of fibrin clots. As relevant models, we utilize a series of low-generation PAMAM dendrimers, including cationic (G2-NH 2 and G4-NH 2 ) and anionic (G3.5-COOH) dendrimers, as well as the clinically relevant hydroxyl-terminated G4 dendrimer (G4-OH). Section title: Reagents Educational score: 2.0385875701904297 Domain: biomedical Document type: Study Language: en PAMAM dendrimers were from Sigma-Aldrich (São Paulo, Brazil). Human fibrinogen (plasminogen-depleted) was from Enzyme Research (South Bend, IN). Human α-thrombin, tPA, and corn trypsin inhibitor (CTI) were from Innovative Research (Novi, MI). Actin FS, Thromborel S, and Dade Innovin were from Siemens Healthineers (Erlangen, Germany). Arachidonic acid was from Sigma-Aldrich (São Paulo, SP, Brazil). The substrate Z-Gly-Gly-Arg-AMC was from Bachem (Torrance, CA). AlexaFluor 488-conjugated fibrinogen was from Merck (São Paulo, Brazil). The following buffer solutions were prepared before each experiment according to standard protocols: TBS (100 mM Tris–HCl, 150 mM NaCl, 0.01% tween 20, pH 7.4), HBS , and HEPES–Tyrode (5 mM HEPES, 137 mM NaCl, 2.9 mM KCl, 12 mM Na 2 HPO 4 , 5 mM C 6 H 12 O 6 , 1 mM CaCl 2 , 1 mM MgCl 2 , pH 7.4). Section title: Blood
Collection Educational score: 4.07728910446167 Domain: biomedical Document type: Study Language: en Blood was drawn from five volunteers following approval from the institution’s Research Ethics Committee. Collection was done in BD Vacutainer tubes (BD Bioscience, São Paulo, Brazil) containing sodium citrate as an anticoagulant. Platelet-poor plasma was prepared from the anticoagulated blood following standard protocols. 52 Aliquots of plasma were stored at −80 °C until use. Before each experiment, plasma samples were thawed in a 37 °C water bath and used immediately, with any remaining volume discarded. Additionally, fresh citrated whole blood from up to seven individual donors was used directly in ROTEM experiments, as detailed in Section 2.12 . Section title: Fibrin
Clot Formation through Optical Turbidimetry Educational score: 4.147921562194824 Domain: biomedical Document type: Study Language: en Purified fibrinogen (4 μM, or 1.4 g/L) in TBS buffer was incubated with dendrimers (1, 10, and 50 μM) for 15 min at room temperature (RT) in a 96-well plate, followed by the addition of thrombin (0.2 NIH/mL) to initiate clotting. Clot formation was monitored at 37 °C over time through turbidity readings at 350 nm using a VersaMax microplate reader (Molecular Devices). From the progress curves, the time to 5% clotting was determined with the “Clotting_or_HaloCL” Shiny app. 53 The impact of dendrimers on the kinetics of clot formation was further evaluated in human plasma. For this purpose, citrated plasma was diluted 1:3 in HBS and incubated with dendrimers (1, 10, and 50 μM) for 30 min at RT. Clot formation was initiated by adding thrombin (0.2 NIH/mL) or the aPTT reagent (25% v/v) with CaCl 2 (10 mM), and then monitored over time by measuring turbidity at 350 nm. All concentrations were final after mixing. Section title: aPTT and PT Assays Educational score: 4.132720947265625 Domain: biomedical Document type: Study Language: en For the aPTT assay, citrated human plasma was incubated with and without dendrimers (1, 10, and 50 μM) in HBS buffer for 10 min at 37 °C. Following this, samples were incubated with Actin FS (25% v/v) for 1 min and then recalcified (10 mM CaCl 2 ). The time to clot formation was monitored using a Dade Behring BFT-II semiautomated coagulation analyzer (Siemens Healthineers). For the PT assay, a similar procedure was followed, except that Thromborel S (50% v/v) was used to activate the coagulation cascade. All concentrations and volume fractions were final after mixing. Section title: Thrombin Generation Assay
(TGA) Educational score: 4.1658782958984375 Domain: biomedical Document type: Study Language: en Citrated human plasma, containing CTI (10 μM) to prevent contact activation of FXII, was incubated with and without dendrimers (1, 10 and/or 50 μM) for 30 min at 37 °C in a 96-well plate. A low concentration of recombinant human TF (Innovin reagent; 1 pM) was used to trigger the coagulation cascade. Then, a premixed solution containing CaCl 2 (10 mM) and the thrombin-specific Z-Gly-Gly-Arg-AMC fluorogenic substrate (420 μM) was added to the well plate immediately before the start of data collection. The total volume in each well was 120 μL, with the plasma volume corresponding to 80 μL. All concentrations were final after mixing. Additional experiments were conducted similarly, except that Actin FS (4.2% v/v) was used to trigger the coagulation cascade in citrated human plasma without CTI. Data collection was carried out on a FlexStation 3 microplate reader (Molecular Devices) using excitation and emission wavelengths of 390 and 460 nm, respectively. Thrombin generation curves were calculated with the “ThrombinCL” Shiny app. 53 Section title: Clot Lysis Educational score: 4.138889312744141 Domain: biomedical Document type: Study Language: en Citrated human plasma diluted 1:3 in HBS buffer was incubated with and without dendrimers (1, 10, and 50 μM) and tPA (1.0 nM) for 30 min at RT in a 96-well plate. The plasma samples were then treated with thrombin (0.4 NIH/mL) and CaCl 2 (10 mM) to initiate clotting. All concentrations were final after mixing. The amount of tPA was adjusted so that clot lysis began only after reaching the same maximum absorbance as in the absence of tPA, indicative of a fully formed clot. Clot formation and lysis were monitored at 37 °C through turbidity readings at 350 nm using a microplate reader. Time to 50% lysis was determined using the “ClotLysisCL” Shiny app. 53 Section title: Wavelength-Dependent Turbidimetry Educational score: 4.192503452301025 Domain: biomedical Document type: Study Language: en Fibrinogen (4 μM) in TBS buffer was loaded into 1 cm wide cuvettes and incubated with and without dendrimers (10 and 50 μM) at RT for 30 min. This was followed by the addition of thrombin (0.4 NIH/mL) to initiate clotting. In another set of experiments, human plasma diluted 1:6 in HBS was incubated with and without dendrimers (1, 10, and 50 μM) at RT for 30 min, followed by the addition of thrombin (0.4 NIH/mL) and CaCl 2 (10 mM) to initiate clotting. Both sets of samples were incubated for an additional 1.5 h to stabilize and consolidate the clots. Absorbance readings were performed using a Shimadzu UV–1800 spectrophotometer, with measurements covering the range from 500 to 800 nm. The determination of fibrin fiber diameter was performed using the corrected Yeromonahos approach, implemented in an Excel spreadsheet provided by Belcher et al. 54 This approach offers the most accurate diameter values, with <20% error within the range of ∼130 to 260 nm. Values of mass-per-length (MPL) ratio were calculated using the Carr-Hermans approach, also implemented in an Excel spreadsheet provided by the same authors. 55 The number of protofibrils per fiber was then calculated by dividing the obtained MPL values by the MPL of a single protofibril (1.44 × 10 11 Da/cm). 56 This approach shows <20% error for fibers up to 200 nm in diameter. It is important to note that these error estimates are based on fibers being sufficiently longer than the wavelength of light. Section title: Laser
Scanning Confocal Microscopy Educational score: 4.133562088012695 Domain: biomedical Document type: Study Language: en Citrated human plasma diluted 1:3 in HBS buffer was spiked with AlexaFluor 488-conjugated fibrinogen (50 nM), making up approximately 0.8–1.6% of the total fibrinogen. The plasma samples were transferred to a 35 mm glass-bottom dish and incubated with and without dendrimers (10 and 50 μM) at RT for 30 min. Subsequently, clot formation was triggered by adding thrombin (0.8 NIH/mL) and CaCl 2 (10 mM), and samples were further incubated at RT in a humidity chamber for 2 h prior to imaging. All concentrations were final after mixing. Clots were imaged using a Zeiss LSM 780 confocal microscope. The confocal data were collected from two independent experiments, each performed in technical duplicate. Approximately 30 optical sections were captured at 0.4 μm intervals using a × 40-magnification oil objective. Control samples lacking thrombin and Ca 2+ were prepared under identical conditions to evaluate if dendrimers induced fibrinogen aggregation independently. Section title: Clot
Permeability Educational score: 4.2023844718933105 Domain: biomedical Document type: Study Language: en Undiluted citrated human plasma was incubated with and without dendrimers (50 and 150 μM) at RT for 30 min. Next, thrombin (0.8 NIH/mL) and CaCl 2 (10 mM) were added to initiate clotting, and a small volume of the mixture (100 μL) was immediately transferred (before clot formation) to a 4.5 cm plastic pipet tip. The samples were kept in a humidity chamber at RT for 2 h to allow full clot formation and stabilization. After this, the plastic tip was joined to a syringe filled with HBS buffer through a silicon tube. The clot was washed with buffer for 1.5 h before sample collection began. Permeation measurements were performed by collecting the volume of buffer passing through the clot under a constant 4 cm pressure drop. Clot permeability was calculated using Darcy’s formula: 1 where Q (cm 3 ) is the volume of buffer collected at a given time t , L is the length of the clot (1.7 cm), η is the liquid viscosity (10 –7 dyne·s·cm –2 ), A is the cross-sectional area of the clot (0.071 cm 2 ), and Δ P is the pressure drop . Section title: Clot Rheometry Educational score: 4.143685817718506 Domain: biomedical Document type: Study Language: en Citrated human plasma diluted 1:2 in HBS buffer was incubated with and without dendrimers (10 and 50 μM) for 30 min at 37 °C. The samples were then transferred to a rheometer (Anton-Paar) and clotting was initiated by adding thrombin (0.4 NIH/mL) and CaCl 2 (10 mM). All concentrations were final after mixing. The storage modulus ( G ′) and loss modulus ( G ′’) during clot formation were monitored over time in oscillatory mode, using a frequency of 1 Hz and a strain (γ) of 1% for 1500 s. Section title: Hemolysis and Platelet Aggregation Educational score: 3.6109235286712646 Domain: biomedical Document type: Study Language: en The general experimental procedure for investigating the influence of dendrimers on red blood cell (RBC) hemolysis and platelet aggregation is outlined in the Supporting Information , and it has also been detailed in previous reports. 57 Section title: Rotational Thromboelastometry Educational score: 4.095440864562988 Domain: biomedical Document type: Study Language: en Citrated human whole blood samples (300 μL) were incubated with G3.5-COOH and G4-OH at 37 °C for 30 min. Blood from three individual donors was used to obtain data for G3.5-COOH at a concentration of 50 μM, while blood from four donors was used for G4-OH at concentrations of 1, 10, and 50 μM. Control samples contained no dendrimers. Coagulation was initiated by adding Actin FS (15% v/v) with CaCl 2 (10 mM) or Thromborel S (15% v/v) with CaCl 2 (10 mM). All concentrations reflect final values after mixing. Samples were analyzed using a four-channel computerized ROTEM system (Pentapharm, Germany). Section title: Statistical Analysis Educational score: 3.211846113204956 Domain: biomedical Document type: Study Language: en Data are presented as mean ± SD. Statistical significance between two groups was assessed using Student’s t test, while comparisons among multiple groups were performed using one-way ANOVA followed by Tukey’s posthoc test. A p -value <0.05 was considered statistically significant. Section title: Results
and Discussion Educational score: 4.06478214263916 Domain: biomedical Document type: Study Language: en We adopted a multilevel characterization approach to assess the potential impact of dendrimers on fibrin clot formation and blood clotting, utilizing samples of purified fibrinogen, human plasma, and human whole blood. The various methods employed are summarized in Figure 1 B. Notably, the chosen set of techniques is commonly used to understand how various health conditions—such as cirrhosis, cardiovascular diseases, clotting factor deficiencies, infections, sepsis, and even exposure to particulate air pollution—influence multiple aspects of blood clotting. 42 − 44 , 46 , 58 − 61 Section title: Results
and Discussion Educational score: 4.065951347351074 Domain: biomedical Document type: Study Language: en To establish a reasonable range of dendrimer concentrations, we took into account the peak plasma concentration of 4 μM for PAMAM G4-OH-based dendrimers reported in a recent human clinical trial. 11 The concentrations we used in our experiments primarily included 1 and 10 μM, which are similar to the peak levels found in vivo, and 50 μM, which is roughly 10 times higher than the peak level. Section title: Results
and Discussion Educational score: 4.119431495666504 Domain: biomedical Document type: Study Language: en Before beginning, we employed optical turbidimetry to ensure that the dendrimers alone did not induce fibrinogen aggregation in samples of purified fibrinogen and citrated human plasma . This outcome differs from a previous study in which a high-generation cationic G7 PAMAM dendrimer was found to cause rapid and extensive fibrinogen aggregation in a thrombin-independent manner. 38 Additionally, we confirmed that none of the dendrimers activated the coagulation cascade at the level of FXII . The observation that anionic G3.5-COOH did not activate FXII contrasts with our previous findings involving anionic usGNPs of a comparable size, which were found to interact with and convert FXII into FXIIa. 50 Overall, these preliminary results indicated that the dendrimers themselves did not produce discernible changes in anticoagulated (citrated) human plasma. Section title: Dynamics of Fibrin Clot Formation Educational score: 4.224829196929932 Domain: biomedical Document type: Study Language: en We employed optical turbidimetry to examine the impact of dendrimers on the dynamics of fibrin clot formation. This method relies on the light scattering caused by fibrin fibers, leading to increased turbidity in the solution over time. Initially, we monitored the clot formation process in a simplified system consisting of purified fibrinogen, using thrombin to initiate clotting. From the progress curves , the time to 5% clotting was extracted and used as a measure of clotting time (CT) . It can be seen that G4-NH 2 drastically accelerated clot formation, with G2-NH 2 exhibiting a similar but less pronounced effect. As a control, we confirmed that G4-NH 2 did not significantly increase thrombin activity relative to the other dendrimers, ruling it out as a factor for the trends observed in Figure 2 . These findings are consistent with those previously reported by Aisina et al. 37 The pronounced acceleration of clotting dynamics by G4-NH 2 is likely the result of fibrin aggregation, as discussed below. G3.5-COOH also shortened CT in a dose-dependent manner; however, unlike G4-NH 2 , this effect was linked to a decrease in maximum turbidity. We note that the outcome for G3.5-COOH contrasts sharply with that for anionic usGNPs of similar size, which significantly delayed fibrinogen clotting. 49 Although the reason for this difference is not yet understood, we note that dendrimers are more flexible structures than usGNPs, which may influence their modes of interaction and binding strength toward fibrinogen. Section title: Dynamics of Fibrin Clot Formation Educational score: 4.111309051513672 Domain: biomedical Document type: Study Language: en Next, we assessed the impact of dendrimers in the more physiological setting of blood plasma. First, we incubated citrated human plasma with dendrimers and added thrombin to trigger clotting. It can be seen that none of the dendrimers had a noticeable impact on clot formation kinetics; however, maximum turbidity increased, particularly for G2-NH 2 and G4-NH 2 . In separate experiments, we used the activated partial thromboplastin time (aPTT) reagent, Actin FS, which contains phospholipids and ellagic acid, to initiate the coagulation cascade at the level of FXII. Under these conditions, G3.5-COOH and G4-OH did not alter the progress curves, whereas both G2-NH 2 and G4-NH 2 significantly delayed the clotting process , with lag times extending to ∼2–25 min depending on the dendrimer type and concentration. Section title: Dynamics of Fibrin Clot Formation Educational score: 4.177176475524902 Domain: biomedical Document type: Study Language: en To complement the above studies, we investigated the impact of dendrimers on clot formation using the standardized aPTT and prothrombin time (PT) assays, which probe the “intrinsic” and “extrinsic” coagulation pathways, respectively. 62 For this purpose, plasma samples containing dendrimers were activated at the level of FXII and TF/factor VIIa using the Actin FS and Thromborel S reagents, respectively. The corresponding CT was then recorded using a mechanical coagulometer. Results for the aPTT assay showed that G3.5-COOH did not change CT beyond the clinically accepted range, while G4-OH increased CT to 55 s when present at a concentration of 50 μM . On the other hand, both cationic dendrimers increased CT even at a lower concentration of 1 μM, and at the highest concentrations, the samples failed to clot within 200 s of measurement . Results from the PT assay indicated that G2-NH 2 , and particularly G4-NH 2 , prolonged CT, whereas G3.5-COOH and G4-OH showed no effect . Our findings for G4-NH 2 are consistent with those reported by Markowicz-Piasecka et al. 34 Section title: Dynamics of Fibrin Clot Formation Educational score: 4.110569477081299 Domain: biomedical Document type: Study Language: en In sum, the above results demonstrate that dendrimers can have varying effects on the dynamics of clot formation depending on the investigated system (purified fibrinogen vs human plasma), dendrimer type and concentration, and measurement method. Most significantly, experiments conducted in human plasma demonstrated that G2-NH 2 and G4-NH 2 significantly delayed the onset of clot formation. However, this effect was observed only when clotting was initiated at the level of FXII or TF, not when triggered by thrombin. This suggests that both dendrimers interfere with coagulation reactions upstream of thrombin, a hypothesis further corroborated in Section 3.3 . Conversely, both G3.5-COOH and G4-OH did not affect the onset of clot formation in human plasma, except for G4-OH at high concentrations in the aPTT assay. Section title: Maximum Turbidity of Fibrin Clots Educational score: 4.165049076080322 Domain: biomedical Document type: Study Language: en The maximum absorbance observed in turbidimetry traces is influenced by several factors, including fibrinogen concentration, the ultrastructure of fibrin fibers (fiber thickness and mass-per-length ratio), and fibrin aggregation. In samples of purified fibrinogen , both G3.5-COOH and G4-OH reduced the maximum turbidity, with G3.5-COOH showing a pronounced effect (see Section 3.4 for further analysis). In contrast, G2-NH 2 and G4-NH 2 increased turbidity, with G4-NH 2 showing a particularly significant change. For both G2-NH 2 and G4-NH 2 , the large increase in maximum turbidity combined with the significant acceleration of CT suggests that these dendrimers actually induced fibrin aggregation in the presence of thrombin. In samples of human plasma activated with the aPTT reagent, both G2-NH 2 and G4-NH 2 appeared to induce fibrin aggregation, as evidenced by the erratic increase in absorbance over time . Section title: Dynamics of Thrombin Generation Educational score: 4.089548110961914 Domain: biomedical Document type: Study Language: en The time course of thrombin generation (TG) in human plasma is a key factor influencing fibrin polymerization and the ensuing clot structure. 63 Hence, dendrimers might affect clot formation and structure by altering TG dynamics. To investigate this, we employed the thrombin generation assay (TGA) to measure real-time TG in the presence of dendrimers. 64 , 65 Section title: Dynamics of Thrombin Generation Educational score: 4.174290657043457 Domain: biomedical Document type: Study Language: en First, we used Actin FS to trigger the intrinsic coagulation pathway through FXII activation. TGA curves are shown in Figure 5 A, while corresponding TG parameters derived from TGA curves are displayed in Supporting Figure S4 . It can be seen that G2-NH 2 significantly impaired TG, specifically prolonging the lag time and time to peak, while decreasing the peak thrombin concentration and the endogenous thrombin potential (area under the curve). The effect of G4-NH 2 was even more drastic, completely inhibiting TG. In contrast, G3.5-COOH had no influence on TG, while G4-OH surprisingly prolonged lag time and time to peak, although only at the highest concentration of 50 μM. Next, we used recombinant TF (Innovin) to trigger the coagulation cascade. A low concentration of TF was employed to probe the intrinsic coagulation pathway through the thrombin-mediated activation of factor XI. 65 TGA curves and derived TG parameters are shown in Figure 5 B and Supporting Figure S5 , respectively. It can be seen that G2-NH 2 and G4-NH 2 reduced peak thrombin concentration and endogenous thrombin potential, with the larger G4-NH 2 having a more pronounced effect. In contrast, G3.5-COOH and G4-OH only mildly affected TG dynamics up to a concentration of 50 μM. These results are partly consistent with those previously reported by Aisina et al. 37 Section title: Dynamics of Thrombin Generation Educational score: 4.194580078125 Domain: biomedical Document type: Study Language: en Taken together, the above findings indicate that both G2-NH 2 and G4-NH 2 significantly interfere with normal TG. This readily accounts for the observed delay in the onset of clot formation as determined by optical turbidimetry and the aPTT and PT assays . The mechanism driving this phenomenon may be partly related to inhibition of factor X activity. 34 For G4-OH, the observed impairment of TG with Actin FS, but not recombinant TF, implies interference within the contact pathway; however, a detailed mechanistic investigation was beyond our scope. Nevertheless, the TGA results for G4-OH are partly consistent with the findings from the aPTT and PT assays, which showed a prolonged CT in the aPTT but not in the PT test . Section title: Dynamics of Thrombin Generation Educational score: 3.9621925354003906 Domain: biomedical Document type: Study Language: en For the experiments described in sections 3.4 through 3.8 , clotting in human plasma was initiated using thrombin and CaCl 2 . This allowed us to assess the effects of dendrimers on fibrin clot structure, properties, and stability without the confounding influence of TG rates. Section title: Fibrin Fiber Diameter and
Protofibril Number
per Fiber Educational score: 4.124990463256836 Domain: biomedical Document type: Study Language: en We applied wavelength-dependent turbidity as a powerful technique for characterizing the ultrastructural properties of fibrin fibers under wet conditions. This method provides parameters such as average fiber diameter, mass-per-length ratio, number of protofibrils per fiber, and protofibril distance within a fiber. 56 Several models are available in the literature to fit turbidimetry data and extract these metrics. Recently, Hudson and colleagues evaluated the available models against scanning electron microscopy and superresolution optical imaging to identify the models and conditions that offer the most accurate measurements. 54 , 55 This work incorporates these recent advancements. Due to the likelihood of some fibrin aggregation in the presence of G2-NH 2 and G4-NH 2 (especially the latter), the following analyses focused solely on G3.5-COOH and G4-OH. Section title: Fibrin Fiber Diameter and
Protofibril Number
per Fiber Educational score: 4.101775169372559 Domain: biomedical Document type: Study Language: en We began by assessing the impact of G3.5-COOH and G4-OH on fiber diameter and the number of protofibrils per fiber in samples of purified fibrinogen. For this purpose, fibrinogen was incubated with dendrimers and then treated with thrombin and Ca 2+ to induce clotting. The results indicated that fiber diameter remained relatively constant at around 175–200 nm in the presence of both dendrimers . However, both dendrimers significantly reduced the number of protofibrils per fiber, with G3.5-COOH having a more pronounced effect. Next, we conducted similar analyses on samples of human plasma . Under these conditions, both fiber diameter and protofibril count remained constant for both dendrimers. Section title: Overall Fibrin Network Structure Educational score: 4.122602462768555 Domain: biomedical Document type: Study Language: en To further characterize the structural characteristics of the fibrin clot, we employed laser scanning confocal microscopy to visualize the overall clot architecture . 47 Human plasma was spiked with fluorescently labeled fibrinogen, incubated with dendrimers, and then treated with thrombin and Ca 2+ to initiate clotting. Except for G4-NH 2 , none of the dendrimers produced notable changes to clot architecture. G4-NH 2 , in contrast, induced local fibrin aggregation, which was especially visible at 50 μM. As a control, none of the dendrimers caused fibrinogen aggregation in the absence of thrombin and Ca 2+ (not shown). Section title: Clot Permeability Educational score: 4.109766006469727 Domain: biomedical Document type: Study Language: en Permeability analysis, represented by the Darcy’s constant, quantifies the flow rate of a liquid as it moves through a fibrin mesh. 66 It provides a key measure of the average pore size and overall density of a fibrin clot. The basic setup of the permeability experiment is depicted in Figure 8 A. First, we verified that control measurements of the Darcy’s constant from plasma samples without dendrimers matched those reported in the literature . 44 , 61 In the presence of dendrimers, we found that clot permeability remained unchanged up to concentrations of 50 μM (not shown). Thus, although G4-NH 2 caused visible structural changes to the clots as depicted in Figure 7 , these changes did not translate into altered permeability under the given experimental conditions. To explore this further, we increased the dendrimer concentration by 3-fold. This adjustment led to a significant increase in clot permeability with G4-NH 2 , but not with other dendrimers , consistent with the trends observed in the confocal results. Section title: Rheological Properties of Plasma Clots Educational score: 3.7489511966705322 Domain: biomedical Document type: Other Language: en Fibrin clots experience shear stress from blood flow within vessels. Consequently, the biomechanical properties of the fibrin network become essential for its function, with abnormal properties contributing to many thrombotic disorders. 26 Section title: Rheological Properties of Plasma Clots Educational score: 4.115161895751953 Domain: biomedical Document type: Study Language: en We applied oscillatory rheometry to measure the key viscoelastic parameters of the fibrin matrix, namely the storage modulus ( G ′), reflecting elasticity/stiffness (reversible deformation), and the loss modulus ( G ′’), reflecting viscosity/plasticity (irreversible deformation). 67 Moreover, we also obtained the loss tangent, defined as tan δ = G ′’/ G ′, which represents the relative plastic component during deformation. For these experiments, human plasma was incubated with dendrimers and treated with thrombin and Ca 2+ to initiate clotting. Figure 9 shows average curves of the G ′ and G ′’ moduli measured during the polymerization process , alongside the average measured values of G ′, G ′’, and tan δ . Although some trends in G ′ changes caused by dendrimers were observed relative to the control, these differences were not statistically significant. Section title: Plasma Clot Fibrinolysis Educational score: 4.212235927581787 Domain: biomedical Document type: Study Language: en Fibrinolysis refers to the slow breakdown of a blood clot to restore normal blood flow. 68 , 69 Dendrimers could disrupt normal fibrinolysis through different mechanisms, including direct binding and inhibition of key fibrinolytic proteins or altering the ultrastructure and overall architecture of fibrin clots. For instance, in purified systems, anionic dendrimers have been reported to significantly inhibit plasminogen activation by tPA, though this effect was observed at much higher dendrimer concentrations than those used here. 37 Understanding the impact of dendrimers on fibrinolysis is important, since enhanced fibrinolysis is associated with an increased tendency for bleeding and delayed or compromised wound healing, whereas impaired fibrinolysis is linked to the development of thrombosis. Section title: Plasma Clot Fibrinolysis Educational score: 4.147930145263672 Domain: biomedical Document type: Study Language: en To investigate the impact of dendrimers on fibrinolysis, we incubated human plasma with dendrimers and tissue plasminogen activator (tPA). Next, we added thrombin and Ca 2+ to initiate clotting, and then monitored clot formation and lysis through optical turbidimetry. The addition of tPA accelerates lysis by speeding up the conversion of plasminogen into plasmin. The results revealed that G4-NH 2 significantly accelerated lysis , which is likely due to alterations in the overall fibrin network structure . Apart from a slight increase in lysis time induced by G2-NH 2 at 1 μM, no additional changes in the dynamics of fibrinolysis were observed. Section title: Hemolysis
and Platelet Aggregation Educational score: 4.098710536956787 Domain: biomedical Document type: Study Language: en We evaluated the potential impact of dendrimers on RBCs and platelets before proceeding with studies using whole blood ( Section 3.10 ). Furthermore, platelets and RBCs are key components of the blood clotting process. Platelets aggregate to form a plug following vascular injury, while also serving as a scaffold to support and accelerate the fibrinogen coagulation process. 21 RBCs, in turn, interact with fibrinogen, are incorporated into whole blood clots, and affect the structure and mechanical properties of these clots. 70 Section title: Hemolysis
and Platelet Aggregation Educational score: 4.104521751403809 Domain: biomedical Document type: Study Language: en We found that G4-NH 2 caused a slight increase in percent hemolysis at 50 μM, while the other dendrimers had no effect . To study the impact of dendrimers on platelet aggregation, we used light transmission aggregometry with platelet-rich human plasma. The results indicated that G4-NH 2 at 50 μM produced some platelet aggregation on its own, while the other dendrimers showed no effect . Moreover, the results revealed that none of the dendrimers inhibited platelet aggregation when this was stimulated by the agonist arachidonic acid . Section title: Rotational
Thromboelastometry in Whole Blood Educational score: 4.154262542724609 Domain: biomedical Document type: Study Language: en We used the ROTEM technique to determine the influence of dendrimers on the coagulation process in human whole blood. This method measures changes in the viscoelastic properties of blood during coagulation. 71 , 72 Key parameters extracted from this technique include: (i) CT, which measures the time from the onset of activation until the start of coagulation; (ii) clot formation time (CFT), which is the time from CT until a clot amplitude of 20 mm, reflecting the speed of clot strengthening and consolidation; (iii) clot polymerization rate (α-angle), which gauges the early rate of fibrin polymerization; (iv) maximum clot firmness (MCF), which measures clot strength in millimeters and reflects the combined effect of platelets, fibrinogen, and other clotting factors; (v) clot lysis index at 30/45/60 min (LI30/45/60), defined as the percentage of MCF remaining at the given time, used to evaluate the extent of clot breakdown or fibrinolysis. Section title: Rotational
Thromboelastometry in Whole Blood Educational score: 4.086990833282471 Domain: biomedical Document type: Study Language: en We characterized the effects of G3.5-COOH and G4-OH in blood samples treated with Actin FS. We excluded the cationic dendrimers from this analysis because they have already been shown to significantly impair clot formation kinetics . The results showed that G3.5-COOH did not alter any of the ROTEM parameters compared to control measurements . Similarly, G4-OH did not affect the CFT, α-angle, MCF, or LI45 parameters. However, G4-OH significantly prolonged CT at 50 μM, but not at lower concentrations. When the coagulation cascade was triggered at the level of TF using Thromborel S, neither G3.5-COOH nor G4-OH altered any of the ROTEM parameters. Section title: Rotational
Thromboelastometry in Whole Blood Educational score: 4.116265773773193 Domain: biomedical Document type: Study Language: en Interestingly, the ROTEM investigations in whole blood were consistent with the aPTT and PT assay findings, which indicated that G3.5-COOH had no effect on CT, while G4-OH at 50 μM prolonged CT in the aPTT but not in the PT assay . Results from the TGA assay also aligned with this overall picture, showing that G4-OH delayed TG when the coagulation cascade was triggered at the level of FXII . Section title: Conclusions Educational score: 4.080568313598633 Domain: biomedical Document type: Study Language: en We investigated the effects of low-generation PAMAM dendrimers on the kinetics of fibrin clot formation, as well as the structure, properties, and stability of these clots. For this purpose, we employed a variety of analytical techniques and methodologies to gather comprehensive information on the clotting process and resulting fibrin clots. This multifaceted approach is crucial because nanomaterials can perturb various stages of the coagulation pathway. On the other hand, relying on a single diagnostic test provides limited insights and an incomplete assessment of the potential harmful effects of nanomaterials. Section title: Conclusions Educational score: 4.109033584594727 Domain: biomedical Document type: Study Language: en Our findings are summarized in Table 1 . They indicate that all dendrimers influenced at least one clotting parameter. Overall, both cationic dendrimers appeared to be unsuitable for in vivo applications, particularly the larger G4-NH 2 . In comparison, G3.5-COOH and G4-OH showed much less impact on the clotting process. However, for clinically relevant G4-OH dendrimers, we obtained a surprising result, in that G4-OH prolonged clotting time not only in human plasma but also in whole blood, although this occurred only at high dendrimer concentrations (50 μM). Interestingly, G3.5-COOH showed potential as a safer option with respect to the clotting process, since they induced minimal alterations across most tested metrics. Section title: Conclusions Educational score: 4.408171653747559 Domain: biomedical Document type: Study Language: en Dissecting the molecular mechanisms underlying the effects described in Table 1 is a complex challenge, as dendrimers may interfere with multiple proteins across the contact, coagulation, and fibrinolytic systems. Nevertheless, it is useful to highlight a few particular aspects related to our findings. (i) For G2-NH 2 , G4-NH 2 , and G4-OH, we found that the prolonged clotting time in human plasma was due to impaired TG, although the mechanism behind this impairment was not investigated. This result is particularly interesting as it contrasts sharply with findings for similarly sized anionic usGNPs, which prolonged clotting time not by interfering with TG but by directly binding to fibrinogen. 49 (ii) We found that G4-NH 2 induced fibrin aggregation in human plasma. This probably accounts for the increased clot permeability/clot porosity associated with these dendrimers. This fibrin aggregation also likely explains the significant reduction in clot lysis time, as the aggregated fibers create more space and facilitate plasmin diffusion within the fibrin mesh. While we also anticipated an impact on the viscoelastic properties of clots treated with G4-NH 2 , we did not observe significant changes (there was a trend toward reduced G ′, but without statistical significance). In contrast, the other dendrimers showed no alterations in fibrin ultrastructural properties (diameter and number of protofibrils) or in the overall architecture of the fibrin mesh. This is in line with the observation that these dendrimers did not affect clot permeability or lysis time. (iii) It is noteworthy that both G3.5-COOH and G4-OH, particularly G3.5-COOH, caused a decrease in maximum turbidity in purified fibrinogen samples. Quantitative analysis revealed that this reduction correlated with a lower number of protofibrils per fiber. This finding suggests that even these more inert dendrimers can modulate aspects of clot architecture. However, in plasma, the presence of other competing dendrimer-protein interactions mitigates these effects, preventing noticeable changes in fibrin clot structure. Section title: Conclusions Educational score: 3.8374478816986084 Domain: biomedical Document type: Study Language: en In summary, our results highlight the potential risks and benefits associated with dendrimer use in clinical settings. More broadly, fundamental studies like this one can provide valuable insights into the inherent biological properties of dendrimers. 73 Understanding how to harness these properties, including the modulatory effects of dendrimers within the contact, coagulation, and fibrinolytic systems, could open new avenues for clinical applications. | Review | biomedical | en | 0.999996 |
Subsets and Splits